Serum-based biomarkers of pancreatic cancer and uses thereof for disease detection and diagnosis

ABSTRACT

Biomarkers of pancreatic cancer are described, as well as methods using these compounds for detecting pancreatic cancer. The methods can be used to diagnose a patient&#39;s health state, or change in health state, or for diagnosing risk of developing or the presence of pancreatic cancer. The method comprises analyzing a sample from a patient to obtain quantifying data for one or more than one of the metabolite markers; comparing the quantifying data to corresponding data obtained for one or more than one reference sample to identify abnormalities in the level of the metabolite marker(s) in the sample; and making a diagnosis if an abnormality is observed. Standards and kits for carrying out the method are also described.

This application a continuation of U.S. patent application Ser. No. 13/499,369, which is a national stage application under 35 U.S.C. § 371 from PCT Application No. PCT/CA2010/001565, filed Oct. 1, 2010, which claims the priority benefit of U.S. Provisional Application No. 61/247,828, filed Oct. 1, 2009.

FIELD OF INVENTION

The present invention relates to biomarkers and methods of detecting diseases and physiological conditions. More specifically, the invention relates to biomarkers of pancreatic cancer and methods using these compounds for detecting diseases and physiological conditions, especially pancreatic cancer.

BACKGROUND OF THE INVENTION

The incidence of pancreatic cancer has increased during the past decades throughout the world, and ranks as the fourth and sixth leading causes of cancer in North America and the European Union respectively (1). This high rank is due to a very poor overall survival (OS) rate (less than 4%), which is illustrated by an annual incidence rate of pancreatic cancer almost identical to the mortality rate. In Canada for example, 3800 new cases were expected to be diagnosed in 2008 with 3700 anticipated deaths from this cancer.

Diagnosis is difficult because there are no noticeable symptoms in early stages, and signs are common with many other illnesses. Furthermore, pancreas location behind other organs renders its imaging more difficult. Diagnosis is usually performed when cancer has already disseminated to other organs. In combination with this late detection, pancreatic cancer displays a poor response to chemotherapy, radiation therapy, and surgery as conventionally used. For patients with advanced pancreatic cancer, the OS rate is less than 1% at five years, whereas for the rare patients diagnosed at an early stage, when surgery is possible, the after resection OS rate climbs to 20% (2). These numbers emphasize the need for an early detection and a new treatment concept of pancreatic cancer.

Current detection methods mostly rely on imaging and are summarized in Table 1.

TABLE 1 Current pancreatic cancer detection methods (adapted from cancer.gov) Imaging Computed Tomography (CT) Scan Ultrasonography Transabdominal Ultrasound Endoscopic Ultrasound Magnetic Resonance Imaging (MRI) Endoscopic Retrograde Cholangiopancreatography Percutaneous Transhepatic Cholangiography Biopsies Fine-Needle Aspiration (FNA) Biopsy Brush Biopsy Laparoscopy Lab tests Bilirubin and other substances

The most sensitive and specific screening tool currently available seems to be the endoscopic ultrasound (3, 4), but its invasive features restrict its use to the screening of high risk populations, namely kindred with minimum two affected first-degree relatives or with known hereditary pancreatic cancer. Another inconvenience of endoscopic ultrasound is that its use is recommended to be associated to other methods such as computed tomography and endoscopic retrograde cholangiopancreatography (5). Diagnosis is confirmed exclusively on analysis of a biopsy. Thus, in addition to being invasive, this multi-step detection and diagnosis process only establishes the presence of an already developed tumor and does not identify risks of developing cancer.

New technologies such as genomics, proteomics, metabolomics and glycomics, have been used in the search for blood-based tumor markers, and have identified glycoproteins, more specifically highly glycosylated mucins, as main tumor markers in all kinds of cancer (6). Among these highly glycosylated mucins, which can be detected by specific monoclonal antibodies, the Cancer Antigen 19-9 (CA 19-9) is present primarily in pancreatic and biliary tract cancers, but also in patients with other malignancies (e.g. colorectal cancer) and benign conditions such as cirrhosis and pancreatitis. CA 19-9 is detected in most protcomics studies in pancreatic cancer serum samples (such as (7)), but its low specificity does not recommend it as a pancreatic cancer biomarker. Anecdotally so far, another glycosylation-related potential biomarker of pancreatic cancer is the core fusylation of biantennary glycans of RNase I, which displayed a 40% increase in the serum of two pancreatic cancer patients relative to two healthy controls (8).

Another well-known serum marker of pancreatic cancer is CEA (carcinoembryonic antigen), with an average reported sensitivity and specificity of both 65% (7). HIP/PAP-I and MIC-1 (macrophage inhibitory cytokine I) are also classical serum markers (9, 10). According to one study, MIC-1 and CA19-9 seem the markers with the highest sensitivity and specificity, in the sense of specificity vs. chronic pancreatitis (and not vs. colon cancer for example), when compared to osteopontin, TIMP-1 and HIP/PAP-I (9).

The use of CA19-9 as a marker is now recommended in combination with other markers, such as the mutation status of pancreatic cancer-related oncogenes like K-ras (2). K-ras is reported to be mutated in 78% of pancreatic adenocarcinomas (11). Molecular events in pancreatic carcinogenesis have been extensively studied (12), and beside K-ras, p53, p21, p16, p27, SMAD4, and cyclin DI are a few of these genes whose mutations or alterations in expression have been associated to pancreatic cancer (12). However, evidence regarding their application as prognostic indicators is conflicting. For instance, there is no consensus on the association between mutation in p53 and decreased survival (12).

MicroRNA profiling has also been performed for pancreatic cancer, with the identification of some common microRNAs specifically altered (13-15).

Protein markers show the advantage of simple screening through an ELISA (Enzyme-linked immunosorbent assay) method, and research in this field is therefore very intensive. Newer proteomics studies have identified additional protein markers, such as apolipoproteins A-I and A-II, and transthyretin (7), all decreased in serum of pancreatic cancer patients, as well as MMP-9, DJ-1 and A1BG, each of which is overexpressed in pancreatic juice from cancer patients (16).

The involvement of apolipoproteins is interesting since they participate in lipid metabolism (17) and other members of this family have been associated to cancer (18).

The fatty acid composition of lipids in plasma and bile from patients with pancreatic cancer has also been analyzed (19, 20), even though neither of these studies has detailed the chemical subfamilies of the altered lipids. Plasma from pancreatic patients showed significantly lower levels of phospholipids that contain the side chain 18:2(ω6), 20:5(ω3) or 22:5(ω3), without distinction of lipid classes (19). Bile from hepatopancreaticobiliary cancer patients was found to contain a much lower level of phosphatidylcholines without distinction of side chains (20).

Since diabetes mellitus (DM) has a high prevalence in pancreatic cancer patients and is frequently of new onset, research has also been aimed at determining whether DM can be utilized as an early pancreatic cancer marker (21). A 2-fold increase of the glucagon/insulin ratio was found in the blood of pancreatic cancer patients relative to healthy controls, and at a cut-off of 7.4 ng/mU glucagon/insulin, pancreatic cancer induced new-onset DM could be discriminated from type 2 DM with 77% sensitivity and 69% specificity (21).

Overall, the methods described above are not ideally suited for large-scale population screening (either for low compliance or low sensitivity and specificity except in the case of a still-to-optimize multiple method combination), and most are capable of detecting pancreatic cancer after the formation of a tumor only. As a result, there still remains a need for accurate methods of detection, particularly for methods to detect early stages of the disease.

SUMMARY OF THE INVENTION

It is an object of the invention to provide diagnostic methods and diagnostic markers useful for detecting cancer in a subject.

Accordingly, the invention relates to methods and diagnostic markers for detecting or diagnosing cancer. Such methods and diagnostic markers are particularly useful for detecting pancreatic cancer.

As an aspect of the invention, a method is provided for diagnosing a subject's pancreatic cancer health state or change in health state, or for diagnosing pancreatic cancer or the risk of pancreatic cancer in a subject, comprising steps of:

-   -   a) analyzing a sample from the patient by high resolution mass         spectrometry to obtain accurate mass intensity data;     -   b) comparing the accurate mass intensity data to corresponding         data obtained from one or more than one reference sample to         identify an increase or decrease in accurate mass intensity; and     -   c) using the increase or decrease in accurate mass intensity for         diagnosing the patient's pancreatic cancer health state, or         change in pancreatic cancer health state, or for diagnosing risk         of developing pancreatic cancer or the presence of pancreatic         cancer in the patient,         wherein the accurate mass intensity is measured, in Daltons, at         or substantially equivalent to a hydrogen and electron adjusted         accurate mass, or neutral accurate mass as described in further         detail herein, for example in Table 5.

In an embodiment, the accurate mass intensity is measured at one or more of the following masses: 78.0516; 84.0575; 112.0974; 116.5696; 191.5055; 197.0896; 200.1389; 202.045; 203.1155; 214.1204; 214.1205; 232.1309; 233.1345; 240.0997; 243.0714; 244.0554; 254.1127; 255.1161; 256.2403; 260.0033; 262.0814; 268.1284; 270.0323; 270.0867; 276.0948; 280.2403; 280.2404; 281.2432; 281.2435; 282.2558; 282.2559; 283.2591; 283.2595; 284.9259; 300.1186; 300.2067; 302.0945; 302.222; 302.2457; 304.2375; 304.2407; 317.9613; 318.0931; 326.2048; 326.2458; 327.9902; 328.2403; 328.2408; 328.2627; 329.2439; 329.2658; 330.2559; 332.1473; 338.0189; 348.1191; 350.2222; 360.1782; 360.1792; 361.1828; 366.3593; 368.1057; 382.1083; 382.1601; 418.2204; 428.2404; 428.3647; 446.2526; 446.3395; 468.2336; 468.3581; 468.3807; 469.237; 469.3616; 481.315; 484.3527; 485.904; 494.4321; 495.3325; 496.3373; 505.3146; 508.2256; 517.3141; 518.321; 519.3295; 520.448; 522.4638; 522.4639; 523.3661; 523.4675; 538.4237; 540.4381; 541.3134; 541.3361; 542.3394; 545.3454; 562.4962; 564.5121; 565.3373; 566.3403; 569.3682; 570.372; 572.4798; 573.4833; 574.4952; 575.4985; 576.4751; 576.5113; 577.5149; 578.5169; 578.5284; 579.5313; 587.3214; 588.3269; 589.3368; 590.3408; 592.4709; 594.4852; 594.4863; 595.4892; 595.4897; 596.5017; 596.5027; 597.5066; 598.4955; 599.4993; 600.5117; 601.5151; 602.5269; 603.5297; 606.5591; 609.3259; 613.3379; 615.3535; 627.5656; 628.5438; 630.799; 631.798; 633.3245; 635.7525; 636.7532; 645.7958; 657.7337; 658.7372; 670.5696; 671.5731; 681.5858; 702.5709; 715.6959; 719.6256; 720.6272; 721.5035; 723.5203; 723.521; 724.5252; 724.5477; 725.7228; 733.5054; 735.6582; 743.5396; 744.5425; 745.5631; 746.5128; 746.5705; 748.527; 749.5374; 749.5388; 750.5425; 751.5511; 751.5539; 752.5574; 755.5497; 757.556; 757.5587; 758.562; 758.5626; 759.5383; 759.5733; 760.5792; 763.5578; 765.5678; 766.4792; 771.5699; 773.5276; 774.5419; 775.5522; 775.5532; 775.5532; 777.0402; 777.5709; 779.5405; 779.5416; 780.5452; 780.5454; 781.5029; 781.5566; 782.5612; 783.569; 783.5755; 784.5742; 784.5806; 785.5913; 785.5929; 785.5931; 786.593; 786.5972; 787.5989; 791.5841; 793.7091; 795.5181; 796.5212; 801.5147; 801.5262; 801.5523; 802.5291; 803.5373; 803.5414; 803.5677; 804.5422; 804.5456; 804.5714; 804.7208; 805.5549; 806.5632; 807.5734; 807.5739; 807.5764; 808.5783; 808.5791; 809.5796; 810.5867; 811.5729; 811.608; 812.6774; 813.5888; 819.5177; 823.5411; 824.69; 825.5522; 826.5561; 826.7047; 827.5401; 827.5678; 827.7082; 828.5397; 828.5721; 829.5516; 829.5532; 829.5843; 830.5591; 830.5879; 831.5652; 831.572; 831.5997; 832.6031; 833.5864; 834.5868; 835.598; 837.7209; 838.7284; 838.7435; 839.7464; 847.531; 850.7061; 850.7326; 851.6694; 851.7107; 851.7337; 852.7368; 853.573; 854.7358; 854.7397; 855.5721; 855.7392; 855.7436; 856.7505; 856.754; 857.6923; 857.7543; 857.7574; 858.7644; 861.749; 865.752; 866.7585; 867.7649; 868.7704; 871.5547; 873.7819; 874.7066; 874.787; 875.7108; 879.7629; 889.7537; 889.8147; 894.7911; 898.7043; 898.7325; 902.7629; 903.7636; 907.7847; 908.7907; 909.7882; 910.7272; 916.7735; 919.6496; 921.813; 922.7081; 922.7285; 922.8222; 923.7295; 924.7233; 925.727; 933.8137; 937.7542; 946.8194; 947.8263; 948.836; 950.7364; 960.7432; 970.733; 972.7481; 973.7482; 984.7406; 986.7568; 996.7518; 997.7397; 998.7566; 999.7632; 1010.765; 1011.669; 1011.77; 1012.781; 1016.931; 1017.935; 1018.944; 1019.951; 1020.957; 1038.915; 1039.705; 1039.921; 1040.933; 1041.935; 1199.084; 1200.088; 1201.09; 1202.098; 1223.09; 1224.096; 1225.096; 1226.599; 1227.112; 1228.117; 1229.12; 1230.125; 1247.084; 1249.105; 1250.108; 1251.119; 1252.12; 1253.123; 1253.134; 1254.137 and 1255.153.

In a further non-limiting embodiment of the invention, the accurate mass intensity is measured at an accurate mass of 519.3295, 523.3661, 541.3134, 702.5709, 724.5477, 757.556, 779.5405, 783.569, 785.5913, 803.5373, 805.5549, 807.5734, 809.5796, 812.6774, 829.5516, 833.5864, 576.4751, 594.4863, 596.5017 or combinations thereof. In such embodiments a decrease in accurate mass intensity is generally identified in the comparing step (b).

In a further exemplary embodiment, the accurate mass is measured at an accurate mass of 600.5117. In such an embodiment an increase in accurate mass intensity is identified in the comparing step (b).

In the above-described method, the term “substantially equivalent” may in certain non-limiting embodiments refer to ±5 ppm of the hydrogen and electron adjusted accurate mass, or neutral accurate mass, and in further embodiments, ±1 ppm of the hydrogen and electron adjusted accurate mass, or neutral accurate mass.

As a further aspect of the invention, there is provided a method for diagnosing a patient's pancreatic cancer health state, or change in pancreatic cancer health state, or for diagnosing risk of developing pancreatic cancer or the presence of pancreatic cancer in a patient, comprising the steps of:

-   -   a) analyzing a sample from the patient to obtain quantifying         data for one or more than one metabolite marker;     -   b) comparing the quantifying data for the one or more than one         metabolite marker to corresponding data obtained for one or more         than one reference sample to identify an increase or decrease in         the level of the one or more than one metabolite marker in the         sample; and     -   c) using the increase or decrease in the level of the one or         more than one metabolite marker in the sample for diagnosing the         patient's pancreatic cancer health state, or change in         pancreatic cancer health state, or for diagnosing risk of         developing pancreatic cancer or the presence of pancreatic         cancer in the patient, wherein the one or more metabolite marker         is as described herein.

In an embodiment, the one or more metabolite marker comprises one or more molecule having a molecular formula as follows: C₃₆H₆₂O₄, C₃₆H₆₂O₅, C₃₆H₆₄O₅, C₃₆H₆₆O₅, C₃₆H₆₄O₆, C₃₆H₆₆O₆, C₃₆H₆₅O₆, C₂₂H₄₆NO₇P, C₂₂H₄NO₇P, C₂₄H₄₆NO₇P, C₂₄H₄₅NO₇P, C₂₄H₄₆NO₇P, C₂₆H₅₄NO₇P, C₂₆H₅₂NO₇P, C₂₆H₅₀NO₇P, C₂₆H₄₅NO₇P, C₂₈H₅₆NO₇P, C₂₈H₅₄NO₇P, C₂₈H₅₂NO₇P, C₂₈H₅₀NO₇P, C₂H₄₅NO₇P, C₂₈H₄₆NO₇P, C₃₀H₃₆NO₇P, C₃₀H₅₄NO₇P, C₃₀H₅₂NO₇P, C₃₀H₈₀NO₇P, C₃₂H₅₈NO₇P, C₃₂H₅₄NO₇P, C₃H₇₆NO₇P, C₄₀H₈₂NO₇P, C₄₀H₈₀NO₇P, C₄₀H₇₈NO₇P, C₄₀H₇₀NO₇P, C₄₂H₇₈NO₈P, C₄₂H₈₀NO₈P, C₄₂H₈₂NO₈P, C₄₂H₈₄NO₈P, C₄₄H₇₈NO₈P, C₄₄H₈₀NO₈P, C₄₄H₈₂NO₈P, C₄₄H₄NO₈P, C₄₄H₈₆NO₈P, C₄₄H₈₄NO₈P, C₄₆H₇₃NO₈P, C₄₆H₈₀NO₈P, C₄₆H₈₂NO₈P, C₄₆H₈₄NO₈P, C₄₈H₈₀NO₈P, C₄₈H₈₂NO₈P, C₄₈H₈₄NO₈P, C₄₈H₈₆NO₈P, C₄₂H₈₀NO₇P, C₄₂H₈₂NO₇P, C₄₂H₈₄NO₇P, C₄₄H₈₂NO₇P, C₄₄H₈₄NO₇P, C₄₄H₈₆NO₇P, C₄₄H₈₈NO₇P, C₄₆H₈₂NO₇P, C₄₆H₈₄NO₇P, C₄₆H₈₆NO₇P, C₄₈H₈₄NO₇P, C₄₈H₈₆NO₇P, C₃₉H₇₉N₂O₆P (or C₃₉H₈₀N₂O₆P⁺), or C₄₁H₈₁N₂O₆P (or C₄₁H₈₁N₂O₆P⁺), or C₄₁H₈₃N₂O₆P (or C₄₁H₈₄N₂O₆P⁺), or C₄₇H₉₃N₂O₆P (or C₄₇H₉₄N₂O₆P), or C₄₇H₉₅N₂O₆P (or C₄₇H₉₆N₂O₆P⁺), including combinations thereof.

In further non-limiting embodiments, the metabolite marker may be a diacylphosphatidylcholine, plasmanylphosphocholine or plasmenylphosphocholine as defined in Formula (I):

including adducts or salts thereof, wherein

-   -   R₁ is a 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5,         22:5 or 22:6 fatty acid or alcohol moiety bonded to the glycerol         backbone, the bond being an acyl linkage when the metabolite         marker is a diacylphosphatidylcholine, an ether linkage when the         metabolite marker is a plasmanylphosphocholine, or a vinyl-ether         linkage when the metabolite marker is a plasmenylphosphocholine;         and     -   R₂ is a 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5,         22:5, or 22:6 fatty acid moiety bonded to the glycerol backbone         through an acyl linkage.

In further embodiments, the metabolite marker may be a 2-lysophosphatidylcholine as defined in Formula (II) or a 1-lysophosphatidylcholine as defined in Formula (III):

-   -   including adducts or salts thereof, wherein     -   R₁ is a 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3,         20:1, 20:2, 20:3, 20:4, 20:5, 20:6, 22:3, 22:4, 22:5, 22:6,         24:4, 24:6, 30:1, 32:0, 32:1, 32:2 or 32:6 fatty acid moiety         bonded to the glycerol backbone through an acyl linkage.

In other non-limiting embodiments, the metabolite marker may be a sphingomyelin as defined in Formula (IV):

-   -   including adducts or salts thereof, wherein the dashed line         represents an optional double bond;     -   R₁ is a C₁₃ alkyl group; and     -   R₂ is a C₁₁ to C₂₅ alkyl or alkenyl group, the alkenyl group         having from 1 to 3 double bonds.

In certain non-limiting embodiments, R₂ of the sphingomyelin of Formula (IV) may be a C₁₁ alkyl group, a C₁₃ alkyl group, a C₁₅ alkyl group, a C₁₇ alkyl group, a C₁₇ alkenyl group with 3 double bonds, a C₁₉ alkyl group, a C₂₁ alkyl group, a C₂₃ alkenyl group with 1 double bond, a C₂₃ alkyl group, a C₂₄ alkyl group, a C₂₅ alkenyl group with 1 double bond, a C₂₅ alkyl group.

The above described methods may further include steps of: analyzing a sample from the patient to obtain quantifying data for one or more than one internal standard molecule; and obtaining a ratio for each of the levels of the one or more than one metabolite marker to the level obtained for the one or more than one internal standard molecule; wherein the comparing step (b) comprises comparing each ratio to one or more corresponding ratios obtained for the one or more than one reference sample.

Without wishing to be limiting in any way, it will be appreciated that the above-described methods can be carried out, at least in part, with the assistance of a computer. In such embodiments the computer may be integrated with the instrument used to perform the analysis, or it may be a separate computer adapted to receive data output from the instrument according to the knowledge and skill of those in the art. The analyzing step (a) will typically be carried out using the instrument, for example but not limited to a mass spectrometer, and the comparing step (b) carried out using the computer or other processing means programmed to receive the accurate mass intensity data or quantifying data from the instrument and perform the calculations required to identify an increase or decrease in the level of the one or more than one metabolite marker in the sample. This data from step (b) may be output for use by an individual trained to identify the noted increase or decrease and make the diagnosis of step (c), or alternatively the computer or processing means may be further programmed to generate an output of a diagnosis. In the latter case, the output may comprise a positive or negative diagnosis factor, and may optionally include additional details including but not limited to statistical data, threshold data, patient data and other details. The data may be output to a display, such as a monitor, to a printer for generating a copy of the details of diagnosis, to a data receiving centre or directly to a service provider, or in any other way as would be understood by one skilled in the art.

In certain embodiments, the metabolite may be a lysophosphatidylcholine (LysoPC), including LysoPC 14:0, LysoPC 14:1, LysoPC 16:0, LysoPC 16:1, LysoPC 16:2, LysoPC 18:0, LysoPC 18:1, LysoPC 18:2, LysoPC 18:3, LysoPC 20:1, LysoPC 20:2, LysoPC 20:3, LysoPC 20:4, LysoPC 20:5, LysoPC 20:6, LysoPC 22:3, LysoPC 22:4, LysoPC 22:5, LysoPC 22:6, LysoPC 24:4, LysoPC 24:6, LysoPC 30:1, LysoPC 32:0, LysoPC 32:1, LysoPC 32:2, LysoPC 32:6, or combinations thereof.

In other embodiments the metabolite may be a phosphatidylcholine, including phosphatidylcholine molecules having a molecular formula of C₄₂H₇₈NO₈P, C₄₂H₈₀NO₈P, C₄₂H₈₂NO₈P, C₄₂H₈₄NO₈P, C₄₄H₇₈NO₈P, C₄₄H₈₀NO₈P, C₄₄H₈₂NO₈P, C₄₄H₈₄NO₈P, C₄₄H₈₆NO₈P, C₄₄H₈₈NO₈P, C₄₆H₇₈NO₈P, C₄₆H₈₀NO₈P, C₄₆H₈₂NO₈P, C₄₆H₈₄NO₈P, C₄₈H₈₀NO₈P, C₄₈H₈₂NO₈P, C₄₈H₈₄NO₈P, C₄₈H₈₆NO₈P, or combinations thereof.

In other embodiments the metabolite may be a plasmenylphosphocholine, including plasmenylphosphocholine molecules having a formula of C₄₂H₈₀NO₇P, C₄₂H₈₂NO₇P, C₄₂H₈₄NO₇P, C₄₄H₈₂NO₇P, C₄₄H₈₄NO₇P, C₄₄H₈₆NO₇P, C₄₄H₈₈NO₇P, C₄₆H₈₂NO₇P, C₄₆H₈₄NO₇P, C₄₆H₈₆NO₇P, C₄₈H₈₄NO₇P, C₄₈H₈₆NO₇P, or combinations thereof.

In yet further embodiments the metabolite may be a sphingomyelin, including sphingomyelin molecules having a molecular formula of C₃₉H₇₉N₂O₆P (or C₃₉H₈₀N₂O₆P⁺), C₄₁H₈₁N₂O₆P (or C₄₁H₈₂N₂O₆P⁺), or C₄₁H₈₃N₂O₆P (or C₄₁H₈₄N₂O₆P⁺), or C₄₇H₉₃N₂O₆P (or C₄₇H₉₄N₂O₆P⁺), or C₄₇H₉₅N₂O₆P (or C₄₇H₉₆N₂O₆P⁺), or combinations thereof.

As described herein, alterations in the levels of the metabolite markers may be detected by MS/MS transition. For instance, a metabolite marker of molecular formula C₃₆H₆₄O₅ may be monitored for level fluctuations of organic extracts in negative ionization mode (such as atmospheric pressure chemical ionization (APCI)) at a MS/MS transition of 575.5/513.5, 575.5/557.5, 575.5/539.5, 575.5/531.5, 575.5/499.5, 575.5/495.5, 575.5/459.4, 575.5/417.4, 575.5/415.3, 575.5/413.3, 575.5/403.3, 575.5/295.2, 575.5/279.2, 575.5/260.2, 575.5/251.2, 575.5/197.9, 575.5/119.4, 575.5/113.1, and 575.5/97.0, or combinations thereof.

Other useful MS/MS transitions for organic extracts in negative ionization mode (e.g. APCI mode) for the metabolite markers described herein include: 593.5/557.5, 593.5/575.4, 593.5/549.4, 593.5/531.5, 593.5/513.4, 593.5/495.4, 593.5/433.3, 593.5/421.4, 593.5/415.2, 593.5/391.4, 593.5/371.3, 593.5/315.3, 593.5/311.1, 593.5/297.2, 593.5/281.2, 593.5/277.2, 593.5/251.2, 593.5/201.1, 593.5/195.3, 593.5/171.1, 593.5/139.1 and 593.5/133.5, or combinations thereof for C₃₆H₆₆O₆; 595.5/559.5, 595.5/577.4, 595.5/551.4, 595.5/533.4, 595.5/515.5, 595.5/497.4, 595.5/478.4, 595.5/433.3, 595.5/423.4, 595.5/391.3, 595.5/372.3, 595.5/595.5/315.3, 595.5/313.2, 595.5/2982, 595.5/297.2, 595.5/281.2, 595.5/279.2, 595.5/239.2, 595.5/232.9, 595.5/171.1, 595.5/169.1 and 595.5/141.1, or combinations thereof for C₃₆H₆₈O₆; 557.4/495.4, 557.4/539.4, 557.4/513.3, 557.4/279.2, 557.4/277.2, 557.4/220.7 and 557.4/111.2, or combinations thereof for C₃₆H₆₂O₄; 573.5/511.4, 573.5/555.3, 573.5/537.4, 573.5/529.4, 573.5/519.4, 573.5/493.3, 573.5/457.4, 573.5/455.3, 573.5/443.4, 573.5/415.4, 573.5/413.3, 573.5/411.3, 573.5/399.3, 573.5/397.3, 573.5/389.7, 573.5/295.2, 573.5/279.2, 573.5/277.2, 573.5/251.2, 573.5/231.1, 573.5/223.1, 573.5/201.1, 573.5/171.1, 573.5/169.1, 573.5/125.1 and 573.5/113.1, or combinations thereof for C₃₆H₆₂O₅; 577.5/515.4, 577.5/559.4, 577.5/546.5, 577.5/533.5, 577.5/497.4, 577.5/419.4, 577.5/405.5, 577.5/297.2 and 577.5/281.2, or combinations thereof for C₃₆H₆₅O₅; 591.5/573.4, 591.5/555.4, 591.5/528.3, 591.5/511.2, 591.5/476.1, 591.5/419.3, 591.5/403.1, 591.5/387.3, 591.5/297.2, 591.5/295.2, 591.5/274.0, 591.5/255.3, 591.5/223.6, 591.5/203.5, 591.5/201.1, 591.5/171.0 and 591.5/125.3, or combinations thereof for C₃₆H₆₄O₆.

Other useful MS/MS transitions for aqueous extracts in positive ionization mode (e.g. positive Electrospray Ionization (ESI)) for the metabolite markers described herein include: 520.3/184.2 for C₂₆H₅₀NO₇P; 524.3/184.2 for C₂₆H₅₄NO₇P; 542.3/184.2 for C₂₈H₄₅NO₇P; 758.6/184.2 for C₄₂H₈₀NO₈P; 784.6/184.2 for C₄₄H₈₂NO₈P; 786.6/184.2 for C₄₄H₈₈NO₈P; 788.6/184.2 for C₄₄H₈₆NO₈P; 790.6/184.2 for C₄₄H₈₈NO₈P; 806.6/184.2 for C₄H₈₀NO₈P; 808.6/184.2 for C₄₆H₈₂NO₈P; 810.6/184.2 for C₄₆H₈₄NO₈P; 834.6/184.2 for C₄₈H₈₄NO₈P; 836.6/184.2 for C₄₈H₈₆NO₈P; 703.6/184.2 for C₃₉H₇₉N₂O₆P; 729.6/184.2 for C₄₁H₈₁N₂O₆P; 731.6/184.2 for C₄₁H₈₃N₂O₆P; 813.6/184.2 for C₄₇H₉₃N₂O₆P; or 815.6/184.2 for C₄₇H₉₅N₂O₆P. Additional MS/MS transition details and other features of the metabolites described herein are evident from the following detailed description of the invention and may also be used in further non limiting embodiments of the invention.

Other useful MS/MS transitions for aqueous extracts in negative ionization mode (e.g. negative ESI) for the metabolite markers described herein include: 564.3/504.3/279.3 for C₂₆H₅₀NO₇P; 568.3/508.4/283.3 for C₂₆H₅₄NO₇P; 586.3/526.3/301.2 for C₂₈H₄₈NO₇P; 802.6/742.6/279.2, 802.6/742.6/281.2, 802.6/742.6/253.2 or 802.6/742.6/255.2 for C₄₂H₈₀NO₈P; 828.6/768.6/305.3, 828.6/768.6/279.2, 828.6/768.6/281.2 or 828.6/768.6/255.2 for C₄₄H₈₂NO₈P; 830.6/770.6/279.2, 830.6/770.6/281.2 or 830.6/770.6/283.2 for C₄₄H₈₄NO₈P; 832.6/772.6/281.2 or 832.6/772.6/283.2 for C₄₄H₈₆NO₈P; 834.6/774.6/283.2 for C₄₄H₈₈NO₈P; 850.6/790.6/327.3, 850.6/790.6/279.2, 850.6/790.6/303.2 or 850.6/790.6/255.2 for C₄₆H₈₀NO₈P; 852.6/792.6/329.3, 852.6/792.6/301.3, 852.6/792.6/303.2, 852.6/792.6/281.2, 852.6/792.6/283.2 or 852.6/792.6/2552 for C₄₆H₈₂NO₈P; 854.6/794.6/331.3, 854.6/794.6/303.2, 854.6/794.6/283.2 or 854.6/794.6/255.2 for C₄₆H₈₄NO₈P; 878.6/818.6/327.3 or 878.6/818.6/283.2 for C₄₈H₈₄NO₈P; 880.6/820.6/329.3 or 880.6/820.6/283.2 for C₄₄H₈₆NO₈P; 747.6/687.6/168.1 for C₃₉H₇₉N₂O₆P; 773.6/713.6/168.1 for C₄₁H₈₁N₂O₆P; 775.6/715.6/168.1 for C₄₁H₈₃N₂O₆P; 857.6/797.6/168.1 for C₄₇H₉₃N₂O₆P; or 859.6/799.6/168.1 for C₄₇H₉₅N₂O₆P. Additional MS/MS transition details and other features of the metabolites described herein are evident from the following detailed description of the invention and may also be used in further non limiting embodiments of the invention.

In the above-described methods, the step of comparing accurate mass intensity data to reference data to identify an increase or decrease in accurate mass intensity; or the step of comparing quantifying data for a metabolite marker to reference data to identify an increase or decrease in the level of the metabolite marker, can in certain non-limiting embodiments comprise or otherwise relate to a step of determining the level of the specified markers, metabolites or molecules, either by determining a change in accurate mass intensity or by other analytical means.

The invention further relates to an assay standard comprising a metabolite marker as described herein labeled with a detection agent. The standard will be useful for carrying out a diagnostic method as described herein, and may include one or more of the following non-limiting detection agents: a stable isotope, an enzyme, or a protein that enables detection in vitro.

In certain non-limiting embodiments, the assay standard may comprise as the metabolite marker a diacylphosphatidylcholine, plasmanylphosphocholine or plasmenylphosphocholine as defined in Formula (I):

-   -   including adducts or salts thereof, wherein     -   R₁ is a 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5,         22:5 or 22:6 fatty acid or alcohol moiety bonded to the glycerol         backbone, the bond being an acyl linkage when the metabolite         marker is a diacylphosphatidylcholine, an ether linkage when the         metabolite marker is a plasmanylphosphocholine, or a vinyl-ether         linkage when the metabolite marker is a plasmenylphosphocholine;         and     -   R₂ is a 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5,         22:5, or 22:6 fatty acid moiety bonded to the glycerol backbone         through an acyl linkage.

In further embodiments, the assay standard may comprise as the metabolite marker a 2-lysophosphatidylcholine as defined in Formula (II) and a 1-lysophosphatidylcholine in Formula (III):

-   -   including adducts or salts thereof, wherein     -   R₁ is a 14:0, 14:1, 16:0, 16:1, 16:2, 18:0, 18:1, 18:2, 18:3,         20:1, 20:2, 20:3, 20:4, 20:5, 20:6, 22:3, 22:4, 22:5, 22:6,         24:4, 24:6, 30:1, 32:0, 32:1, 32:2 or 32:6 fatty acid moiety         bonded to the glycerol backbone through an acyl linkage.

In other non-limiting embodiments, the assay standard may comprise as the metabolite marker a sphingomyelin as defined in Formula (IV):

-   -   including adducts or salts thereof, wherein the dashed line         represents an optional double bond,     -   R₁ is a C₁ alkyl group; and     -   R₂ is a C₁₁ to C₂₅ alkyl or alkenyl group, the alkenyl group         having from 1 to 3 double bonds.

In certain non-limiting embodiments, R₂ of the sphingomyelin of Formula (IV) may be a C₁₁ alkyl group, a C₁₃ alkyl group, a C₁₅ alkyl group, a C₁₇ alkyl group, a C₁₇ alkenyl group with 3 double bonds, a C₁₉ alkyl group, a C₂₁ alkyl group, a C₂₃ alkenyl group with 1 double bond, a C₂₃ alkyl group, a C₂₄ alkyl group, a C₂₅ alkenyl group with 1 double bond, or a C₂₅ alkyl group.

In further embodiments of the standard, which are also considered to be non-limiting, the assay standard may comprise as the metabolite marker a lysophosphatidylcholine (LysoPC, either 1-LysoPC or 2-LysoPC) including LysoPC 14:0, LysoPC 14:1, LysoPC 16:0, LysoPC 16:1, LysoPC 16:2, LysoPC 18:0, LysoPC 18:1, LysoPC 18:2, LysoPC 18:3, LysoPC 20:1, LysoPC 20:2, LysoPC 20:3, LysoPC 20:4, LysoPC 20:5, LysoPC 20:6, LysoPC 22:3, LysoPC 22:4, LysoPC 22:5, LysoPC 22:6, LysoPC 24:4, LysoPC 24:6, LysoPC 30:1, LysoPC 32:0, LysoPC 32:1, LysoPC 32:2, or LysoPC 32:6.

The invention further relates to a kit or commercial package comprising the above-described standard and instructions for quantitating an analyte or performing a diagnostic test as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent from the following description in which reference is made to the following figures.

FIG. 1 provides a schematic description of the studies performed.

FIG. 2 illustrates a Principal Component Analysis on all masses differentiating pancreatic cancer from controls with p-value<0.05 showing a clear separation between pancreatic cancer samples (grey) and controls (black).

FIG. 3 illustrates a Principal Component Analysis on the 20 best biomarkers showing a clear separation between pancreatic cancer samples (grey) and controls (black) (a), and the relative level intensities of these 20 biomarkers in the serum of pancreatic cancer patients relative to controls (b).

FIG. 4 illustrates a ROC and variability chart for the first six best biomarkers by FTICR, namely 594.4863 (AUC=0.96) (a), 785.5913 (AUC=0.93) (b), 702.5709 (AUC=0.91) (c), 807.5734 (AUC=0.93) (d), 576.4751 (AUC=0.93) (e) and 541.3134 (AUC-0.92) (f).

FIG. 5 shows a logistic regression analysis of the combination of the six FTICR best biomarkers, with ROC curve (a) and classification table (b).

FIG. 6 illustrates the fragmentation pattern of C36 compound “576”.

FIG. 7 illustrates the fragmentation pattern of C36 compound “594”.

FIG. 8 illustrates the fragmentation pattern of C36 compound “596”.

FIG. 9 illustrates the fragmentation pattern of C36 compound “558”.

FIG. 10 illustrates the fragmentation pattern of C36 compound “574”.

FIG. 11 illustrates the fragmentation pattern of C36 compound “578” FIG. 12 illustrates the fragmentation pattern of C36 compound “592”.

FIG. 13 shows the ¹H NMR spectrum of the fraction rich in C36 markers “594” and “596”.

FIG. 14 illustrates the fragmentation patterns of 519.3 in positive aqueous ESI mode. (a) and (b) correspond to the fragmentation patterns at different retention times.

FIG. 15 illustrates the fragmentation patterns of 523.3 in positive aqueous ESI mode. (a) and (b) correspond to the fragmentation patterns at different retention times.

FIG. 16 illustrates the fragmentation patterns of 541.3 in positive aqueous ESI mode. (a), (b), (c) and (d) correspond to fragmentation patterns at different retention times.

FIG. 17 illustrates the fragmentation pattern of 757.6 in positive aqueous ESI mode.

FIG. 18 illustrates the fragmentation pattern of 779.5 in positive aqueous ESI mode.

FIG. 19 illustrates the fragmentation pattern of 783.6 in positive aqueous ESI mode, showing three retention times with choline fragments (a), (b), (c).

FIG. 20 illustrates the fragmentation pattern of 785.6 in positive aqueous ESI mode.

FIG. 21 illustrates the fragmentation pattern of 803.5 in positive aqueous ESI mode.

FIG. 22 illustrates the fragmentation pattern of 805.6 in positive aqueous ESI mode.

FIG. 23 illustrates the fragmentation pattern of 807.6 in positive aqueous ESI mode showing two retention times with choline fragments (a), (b).

FIG. 24 illustrates the fragmentation pattern of 809.6 in positive aqueous ESI mode.

FIG. 25 illustrates the fragmentation pattern of 829.6 in positive aqueous ESI mode.

FIG. 26 illustrates the fragmentation pattern of 833.6 in positive aqueous ESI mode.

FIG. 27 illustrates the fragmentation pattern of “757.6” as a formic acid adduct in negative aqueous ESI mode, showing two main side chains, 16:0 (m/z 255.2) and 18:2 (m/z 279.2). “757.6” is therefore PtdCho 16:0/18:2 and PtdCho 18:2/16:0.

FIG. 28 illustrates the fragmentation pattern of “779.6” as a formic acid adduct in negative aqueous ESI mode, showing the side chains 16:0 (m/z 255.2), 20:5 (m/z 301.2) and 20:4 (m/z 303.2) as the most abundant. “779.6” is therefore mostly PtdCho 16:0/20:5, PtdCho 20:5/16:0 and PtdCho 18:2/20:4.

FIG. 29 illustrates the fragmentation pattern of “783.6” as a formic acid adduct in negative ESI aqueous mode, showing the side chains 20:3 (m/z 305.2), 18:2 (m/z 279.2), 18:1 (m/z 281.2) and 16:0 (m/z 255.2) as the most abundant. “783.6” therefore mostly is PtdCho 16:0/20:3 and PtdCho 18:1/18:2.

FIG. 30 illustrates the fragmentation pattern of “785.6” as a formic acid adduct in negative aqueous ESI mode, showing two side chains, 18:0 (m/z 283.3) and 18:2 (m/z 279.2) in one pattern (a) and one main side chain, 18:1 (m/z 281.2) in the other (b). “785.6” is therefore PtdCho 18:0/18:2 and PtdCho 18:1/18:1.

FIG. 31 illustrates the fragmentation pattern of “805.6” as a formic acid adduct in negative aqueous ESI mode at different retention times (a-d). The different side chains, 16:0 (m/z 255.2), 22:6 (m/z 327.3), 18:2 (m/z 279.3) and 20:4 (m/z 303.2), identify “805.6” as PtdCho 22:6/16:0 and, PtdCho 20:4/18:2).

FIG. 32 illustrates the fragmentation patterns of “807.6” as a formic acid adduct in negative aqueous ESI mode at different retention times (a-c). The different side chains, 18:0 (m/z 283.2), 20:5 (m/z 301.2), 16:0 (m/z 255.2), 22:5 (m/z 329.3), 18:1 (m/z 281.3) and 20:4 (m/z 303.2) identify “807.6” as PtdCho 18:0/20:5, PtdCho 16:0/22:5, PtdCho 22:5/16:0 and PtdCho 18:1/20:4.

FIG. 33 illustrates the fragmentation pattern of 702.6 in positive aqueous ESI mode.

FIG. 34 illustrates the fragmentation pattern of 812.7 in positive aqueous ESI mode.

FIG. 35 illustrates the fragmentation pattern of 724.6 in positive aqueous ESI mode

FIG. 36 illustrates the fragmentation pattern of 702.6 as a formic acid adduct in negative ESI analysis mode in control sample aqueous extracts (m/z 747.6).

FIG. 37 illustrates the fragmentation pattern of synthetic SM(d18:1/16:0) (from Avanti Polar Lipids, cat. 860584) as a formic acid adduct in negative ESI analysis mode (m/z 747.6).

FIG. 38 illustrates the fragmentation pattern of 812.7 as a formic acid adduct in negative ESI analysis mode in control sample aqueous extracts (m/z 857.7).

FIG. 39 illustrates the fragmentation pattern of synthetic SM(d18:1/24:1(15Z)) (from Avanti Polar Lipids, cat. 860593) as a formic acid adduct in negative ESI analysis mode (m/z 857.7).

FIG. 40 illustrates the fragmentation of 600.5117 organic extract in positive APCI.

FIG. 41 shows the relative levels of LysoPC18:0 (mass 523.4), LysoPCl8:2 (mass 519.3) and LysoPC20:5 (mass 541.3) and of additional LysoPC in the serum of pancreatic cancer patients relative to controls by Electrospray Ionization (ESI) analysis. (a) LysoPC with 14, 16 and 18 carbons on the side chain, (b) LysoPC with 20, 22 and 24 carbons on the side chain, (c) LysoPC with 30 and 32 carbons on the side chain, and (d) LysoPC with 14, 16, 18, 20 and 22 carbons on the side chain. (a) to (c) in positive ESI analysis mode and (d) in negative ESI analysis mode.

FIG. 42 shows the relative MRM levels of 13 PtdCho named by their parent mass in positive ESI analysis mode (a) 27 PtdCho in negative ESI mode (b), and 12 PlsCho named by their parent mass in positive ESI mode (c) in the serum of pancreatic cancer patients relative to controls.

FIG. 43 shows the relative MRM levels of five sphingomyelins in the serum of pancreatic cancer patients relative to controls.

FIG. 44 shows the relative levels of C36 markers in the serum of pancreatic cancer patients relative to controls.

FIG. 45 shows the relative intensities of biomarkers for pancreatic cancer at different stages in three LysoPC (a), seven PtdCho (b), five sphingomyelins (c) and three C36 markers (d).

FIG. 46 shows the relative intensities of biomarkers for pancreatic cancer chemoradiation therapy status in three LysoPC (a), seven PtdCho (b), five sphingomyelins (c) and three C36 markers (d).

DETAILED DESCRIPTION

The present inventors have identified cancer-specific biomarkers in human serum, and accordingly present herein a non-invasive cancer detection method that is useful for monitoring an individual's susceptibility to disease, and that may be used either alone or in combination with other known diagnostic methods. The methods described are particularly useful for detecting or diagnosing pancreatic cancer.

A “non-targeted” approach was developed for the identification of biomarkers specific to pancreatic cancer. This discovery platform incorporated the use of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which is capable of detecting ions with mass accuracy below 1 part per million (ppm). Using this method, liquid sample extracts can be directly infused, for instance using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), without chromatographic separation. Ions with differing mass to charge (M/Z) ratios are then simultaneously resolved using a Fourier transformation. This combination of liquid extraction, flow injection, high resolution and informatics affords a unique opportunity to broadly characterize the biochemical composition of samples without apriori knowledge.

When analyzing the serum metabolomic profiles of pancreatic cancer patients and healthy asymptomatic subjects included in their study, the inventors identified specific biomarkers that had significantly altered serum levels in pancreatic cancer patients when compared to controls in a set of 90 samples. Structural characterization was performed by MS/MS technology, and some of the markers were found to be choline-related compounds. Alterations in the serum levels of these biomarkers were confirmed by targeted mass spectrometry using a targeted high-throughput triple-quadrupole MRM (TQ-MRM) method on the same samples.

The inventors have accordingly developed methods to monitor levels of these biomarkers in a subject in a specific and sensitive manner, and to use this information as a useful tool for the early detection and screening of pancreatic cancer.

The present invention accordingly relates to a method of diagnosing cancer by measuring the levels of specific biomarkers present in human serum and comparing them to “normal” reference levels. The described method may be used for the early detection and diagnosis of cancer as well as for monitoring the effects of treatment on cancer patients.

The method also may be incorporated into a high-throughput screening method for testing large numbers of individuals, and further enables longitudinal screening throughout the lifetime of a subject to assess risk and detect disease early on. The method therefore has the potential to detect disease progression prior to that detectable by conventional methods, which is critical to positive treatment outcome.

According to the described method, biological samples taken from one or more subjects of a particular health-state category are compared to the same samples taken from the normal population to identify differences in the levels of the described biomarkers. The samples are extracted and analyzed using various analytical platforms including, but not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FTMS) and liquid chromatography mass spectrometry (LC-MS).

The biological samples could originate from anywhere within the body, for example but not limited to, blood (serum/plasma), cerebral spinal fluid (CSF), bile, urine, stool, breath, saliva, or biopsy of any solid tissue including tumor, adjacent normal, smooth and skeletal muscle, adipose tissue, liver, skin, hair, brain, kidney, pancreas, lung, colon, stomach, or other. Of particular interest are samples that are serum or CSF. While the term “serum” is used herein, those skilled in the art will recognize that plasma or whole blood or a sub-fraction of whole blood may be used.

When a blood sample is drawn from a patient there are several ways in which the sample can be processed. The range of processing can be as little as none (i.e. frozen whole blood) or as complex as the isolation of a particular cell type. The most common and routine procedures involve the preparation of either serum or plasma from whole blood. All blood sample processing methods, including spotting of blood samples onto solid-phase supports, such as filter paper or other immobile materials, are also contemplated by the invention.

Without wishing to be limiting, the processed blood or plasma sample described above may then be further processed to make it compatible with the methodical analysis technique to be employed in the detection and measurement of the metabolites contained within the processed blood sample. The types of processing can range from as little as no further processing to as complex as differential extraction and chemical derivatization. Extraction methods may include sonication, soxhlet extraction, microwave assisted extraction (MAE), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), pressurized liquid extraction (PLE), pressurized hot water extraction (PHWE) and/or surfactant assisted extraction (PHWE) in common solvents such as methanol, ethanol, mixtures of alcohols and water, or organic solvents such as ethyl acetate or hexane. A method of particular interest for extracting metabolites for FTMS non-targeted analysis and for flow injection LC-MS/MS analysis is to perform a liquid/liquid extraction whereby non-polar metabolites dissolve in an organic solvent and polar metabolites dissolve in an aqueous solvent.

The extracted samples may be analyzed using any suitable method including those known in the art. For example, and without wishing to be limiting, extracts of biological samples are amenable to analysis on essentially any mass spectrometry platform, either by direct injection or following chromatographic separation. Typical mass spectrometers are comprised of a source that ionizes molecules within the sample, and a detector for detecting the ionized molecules or fragments of molecules. Non-limiting examples of common sources include electron impact, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photo ionization (APPI), matrix assisted laser desorption ionization (MALDI), surface enhanced laser desorption ionization (SELDI), and derivations thereof. Common mass separation and detection systems can include quadrupole, quadrupole ion trap, linear ion trap, time-of-flight (TOF), magnetic sector, ion cyclotron (FTMS), Orbitrap, and derivations and combinations thereof. The advantage of FTMS over other MS-based platforms is its high resolving capability that allows for the separation of metabolites differing by only hundredths of a Dalton, many of which would be missed by lower resolution instruments.

By the term “metabolite”, it is meant specific small molecules, the levels or intensities of which are measured in a sample, and that may be used as markers to diagnose a disease state. These small molecules may also be referred to herein as “metabolite marker”, “metabolite component”, “biomarker”, or “biochemical marker”.

The metabolites are generally characterized by their accurate mass, as measured by mass spectrometry technique. The accurate mass may also be referred to as “accurate neutral mass” or “neutral mass”. The accurate mass of a metabolite is given herein in Daltons (Da), or a mass substantially equivalent thereto. By “substantially equivalent thereto”, it is meant that a +/−5 ppm difference in the accurate mass would indicate the same metabolite. The accurate mass is given as the mass of the neutral metabolite. During the ionization of the metabolites, which occurs during analysis of the sample, the metabolite will cause either a loss or gain of one or more hydrogen atoms and a loss or gain of an electron. This changes the accurate mass to the “ionized mass”, which differs from the accurate mass by the mass of hydrogen atoms and electrons lost or gained during ionization. Unless otherwise specified, the accurate neutral mass will be referred to herein.

Similarly, when a metabolite is described by its molecular formula, the molecular formula of the neutral metabolite will be given. Naturally, the molecular formula of the ionized metabolite will differ from the neutral molecular formula by the number of hydrogen atoms lost or gained during ionization or due to the addition of a non-hydrogen adduct ion.

Data is collected during analysis and quantifying data for one or more than one metabolite is obtained. “Quantifying data” is obtained by measuring the levels or intensities of specific metabolites present in a sample.

The quantifying data is compared to corresponding data from one or more than one reference sample. The “reference sample” is any suitable reference sample for the particular disease state. For example, and without wishing to be limiting in any manner, the reference sample may be a sample from a control individual, i.e., a person not suffering from cancer with or without a family history of cancer (also referred to herein as a “‘normal’ counterpart”); the reference sample may also be a sample obtained from a patient clinically diagnosed with cancer. As would be understood by a person of skill in the art, more than one reference sample may be used for comparison to the quantifying data. For example and without wishing to be limiting, the one or more than one reference sample may be a first reference sample obtained from a non-cancer control individual. In the case of monitoring a subject's change in disease state, the reference sample may include a sample obtained at an earlier time period either pre-therapy or during therapy to compare the change in disease state as a result of therapy.

An “internal control metabolite” refers to an endogenous metabolite naturally present in the patient. Any suitable endogenous metabolite that does not vary over the disease states can be used as the internal control metabolite.

Use of a ratio of the metabolite marker to the internal control metabolite offers measurement that is more stable and reproducible than measurement of absolute levels of the metabolite marker. As the internal control metabolite is naturally present in all samples and does not appear to vary significantly over disease states, the sample-to-sample variability (due to handling, extraction, etc) is minimized.

As discussed above the biomarkers described herein were identified by a method known as non-targeted analysis. Non-targeted analysis involves the measurement of as many molecules in a sample as possible, without any prior knowledge or selection of the components prior to the analysis (see WO 01/57518, published Aug. 9, 2001). Therefore, the potential for non-targeted analysis to discover novel metabolite biomarkers is high versus targeted methods, which detect a predefined list of molecules. The present inventors used a non-targeted method to identify metabolite components that differ between cancer-positive and healthy individuals, followed by the development of a high-throughput targeted assay for a subset of the metabolites identified from the non-targeted analysis.

According to this analysis small molecules, metabolites, or metabolite fragments were identified that have differential abundances between cancer-positive serum and normal serum. As listed in Table 5, the inventors found 362 metabolite masses to have statistically significant differential abundances between cancer-positive serum and normal serum. All of these features, which differ statistically between the two populations, have potential diagnostic utility. However, the incorporation of 362 signals into a commercially diagnostic assay is in many cases impractical, so an optimum diagnostic set of markers or metabolites may be selected, for instance in a panel for a high-throughput screening (HTS) assay.

There are multiple types of HTS assay platform options currently available depending on the molecules being detected. These include, but are not limited to, colorimetric chemical assays (UV, or other wavelength), antibody-based enzyme-linked immunosorbant assays (ELISAs), chip-based and polymerase-chain reaction for nucleic acid detection assays, bead-based nucleic-acid detection methods, dipstick chemical assays, image analysis such as MRI, petscan, CT scan, and various mass spectrometry-based systems.

In a non-limiting embodiment, the HTS assay is based upon conventional triple-quadrupole mass spectrometry technology. The HTS assay works by directly injecting a serum extract into the triple-quad mass spectrometer, which then individually isolates each of the parent molecules by single-ion monitoring (SIM). This is followed by the fragmentation of each molecule using an inert gas (called a collision gas, collectively referred to as collision-induced dissociation or CID). The intensity of a specific fragment from each parent biomarker is then measured and recorded, through a process called multiple-reaction monitoring (MRM). In addition, an internal standard molecule is also added to each sample and subjected to fragmentation as well. This internal standard fragment should have the same intensity in each sample if the method and instrumentation is operating correctly. When all biomarker fragment intensities, as well as the internal standard fragment intensities are collected, a ratio of the biomarker to IS fragment intensity is calculated, and the ratio log-transformed. The values for each patient sample are then compared to a previously determined distribution of disease-positive and controls, to determine the relative likelihood that the person is positive or negative for the disease.

A commercial method for screening patients for cancer using the described assay methods is also envisioned. There are numerous options for the deployment of the assay world-wide. These include, but are not limited to: 1, the development of MS/MS methods compatible with current laboratory instrumentation and triple-quadrupole mass spectrometers which are readily in place in many labs around the world, and/or 2, the establishment of a testing facility where samples could be shipped and analyzed at one location, and the results sent back to the patient or patient's physician.

Structural elucidation of the identified metabolites was carried out using a series of physical and chemical property investigations. The principal characteristics that are normally used for this identification are accurate mass and molecular formula determination, polarity, acid/base properties, NMR spectra, and MS/MS or MSn spectra.

One group of diagnostic biomarkers, referred to herein as the C36 markers (558.4, 574.5, 576.5, 578.5, 592.5, 594.5, 596.5), were determined to have the following molecular formulae, respectively: C₃₆H₆₂O₄, C₃₆H₆₂O₅, C₃₆H₆₂O₅, C₃₆H₆₆O₅, C₃₆H₆₄O₆, C₃₆H₆₆O₆, and C₃₆H₆₈O₆. MS/MS transitions for each of these biomarkers for organic extracts in negative APCI were observed as follows: C₃₆H₆₂O₄: 557.4/495.4, 557.4/539.4, 557.4/513.3, 557.4/279.2, 557.4/277.2, 557.4/220.7 and 557.4/111.2; C₃₆H₆₂O₅: 573.5/511.4, 573.5/555.3, 573.5/537.4, 573.5/529.4, 573.5/519.4, 573.5/493.3, 573.5/457.4, 573.5/455.3, 573.5/443.4, 573.5/415.4, 573.5/413.3, 573.5/411.3, 573.5/399.3, 573.5/397.3, 573.5/389.7, 573.5/295.2, 573.5/279.2, 573.5/277.2, 573.5/251.2, 573.5/231.1, 573.5/223.1, 573.5/201.1, 573.5/171.1, 573.5/169.1, 573.5/125.1 and 573.5/113.1; C₃₆H₆₄O₅: 575.5/513.5, 575.5/557.5, 575.5/531.5, 575.5/499.5, 575.5/495.4, 575.5/447.3, 575.5/417.4, 575.5/415.4, 575.5/413.3, 575.5/371.3, 575.5/295.2, 575.5/279.2, 575.5/260.2, 575.5/251.2, 575.5/459.4, 575.5/403.3, 575.5/197.9, 575.5/119.4, 575.5/113.1, 575.5/97.0 and 575.5/539.5; C₃₆H₆₆O₅: 577.5/515.4, 577.5/559.4, 577.5/546.5, 577.5/533.5, 577.5/497.4, 577.5/419.4, 577.5/405.5, 577.5/297.2 and 577.5/281.2; C₃₆H₆₄O₆: 591.5/573.4, 591.5/555.4, 591.5/528.3, 591.5/511.2, 591.5/476.1, 591.5/419.3, 591.5/403.1, 591.5/387.3, 591.5/297.2, 591.5/295.2, 591.5/274.0, 591.5/255.3, 591.5/223.6, 591.5/203.5, 591.5/201.1, 591.5/171.0 and 591.5/125.3; C₃₆H₆₆O₆: 593.5/557.5, 593.5/513.4, 593.5/495.4, 593.5/371.3, 593.5/315.3, and 593.5/277.2; C₃₄H₆₈O₆: 595.5/577.5, 595.5/559.5, 595.5/551.5, 595.5/549.7, 595.5/533.5, 595.5/279.2, 595.5/391.3, 595.5/515.4, 595.5/478.4, 595.5/423.4, 595.5/372.5, 595.5/315.3, 595.5/313.2, 595.5/433.3, 595.5/298.2, 595.5/239.2, 595.5/232.9, 595.5/171.1, 595.5/169.1, 595.5/141.1 and 595.5/497.4.

A second group of choline-related diagnostic biomarkers, including lysophosphatidylcholines, phosphatidylcholines and sphingomyelins were also identified. The lysophosphatidylcholines include: LysoPC 14:0; LysoPC 14:1; LysoPC 16:0; LysoPC 16:1; LysoPC 16:2; LysoPC 18:0; LysoPC 18:1; LysoPC 18:2; LysoPC 18:3; LysoPC 20:1; LysoPC 20:2; LysoPC 20:3; LysoPC 20:4; LysoPC 20:5; LysoPC 20:6; LysoPC 22:3; LysoPC 22:4; LysoPC 22:5; LysoPC 22:6; LysoPC 24:4; LysoPC 24:6; LysoPC 30:1; LysoPC 32:0; LysoPC 32:1; LysoPC 32:2; and LysoPC 32:6. The molecular weight, formulae and MS/MS transitions for each of these biomarkers are described in further detail below.

The phosphatidylcholines (755.55; 757.56; 759.58; 761.59; 779.54; 781.56; 783.58; 785.59; 787.61; 803.54; 805.56; 807.58; 809.59; 829.55; 831.58; and 833.59), were determined to have the following molecular formulae, respectively: C₄₂H₇₈NO₈P; C₄₂H₈₀NO₈P; C₄₂H₈₂NO₈P; C₄₂H₈₄NO₈P; C₄₄H₇₈NO₈P; C₄₄H₈₀NO₈P; C₄₄H₂NO₈P; C₄₄H₈₄NO₈P; C₄₄H₈₆NO₈P; C₄₆H₇₈NO₈P; C₄₆H₈₀NO₈P; C₄₆H₈₂NO₈P; C₄₆H₈₄NO₈P; C₄₈H₈₀NO₈P; C₄₈H₈₂NO₈P; and C₄₈H₈₄NO₈P. The molecular weight, formulae and MS/MS transitions for each of these biomarkers are described in further detail below.

The sphingomyelins 702.57 and 812.68 were determined to have the respective formulae C₃₉H₇₂N₂O₆P and C₄₇H₉₃N₂O₆P. The molecular weight, formulae and MS/MS transitions for each of these biomarkers are described in further detail below.

The present invention is further defined with reference to the following examples that are not to be construed as limiting.

EXAMPLES Materials & Methods: 1. Patient Sample Selection

Clinical samples were obtained from Osaka Medical University, Japan. Samples were collected, processed and stored in a consistent manner by teams of physicians. All samples were properly consented and were accompanied by detailed pathology reports.

The samples included 50 controls and 40 pancreatic cancer patients, among them 20 had undergone chemoradiation therapy (CRT) and 20 had not at the time of sampling. Four patients were in stage I, four in stage II, five in stage III, 16 in stage IVa and 11 in stage IVb (Table 2).

TABLE 2 Clinical characteristics of the studied population. Stage 1 Stage II Stage III Stage IVa Stage IVb CRT 4 2 2 7 5 no CRT 0 2 3 9 6

All samples were processed and analyzed in a randomized manner and the results unblinded following analysis.

2. Sample Extraction

Serum samples were stored at −80° C. until thawed for analysis, and were only thawed once. All extractions were performed on ice. Serum samples were prepared for FTICR-MS analysis by first sequentially extracting equal volumes of serum with 1% ammonium hydroxide and ethyl acetate (EtOAc) in the ratio of 1:1:5 respectively three times. Samples were centrifuged between extractions at 4° C. for 10 min at 3500 rpm, and the organic layer removed and transferred to a new tube (extract A). After the third EtOAc extraction, 0.33% formic acid was added, followed by two more EtOAc extractions. Following the final organic extraction, the remaining aqueous component was further extracted twice with water, and protein removed by precipitation with 3:1 acetonitrile (extract B). A 1:5 ratio of EtOAc to butanol (BuOH) was then evaporated under nitrogen to the original BuOH starting volume (extract C). All extracts were stored at −80° C. until FTICR-MS analysis.

3. FICR-MS Analysis

Extracts were diluted either three or six-fold in methanol:0.1% (v/v) ammonium hydroxide (50:50, v/v) for negative ionization modes, or in methanol:0.1% (v/v) formic acid (50:50, v/v) for positive ionization modes. For APCI, sample extracts were directly injected without diluting. All analyses were performed on a Bruker Daltonics APEX m Fourier transform ion cyclotron resonance mass spectrometer equipped with a 7.0 T actively shielded superconducting magnet (Bruker Daltonics, Billerica, Mass.). Samples were directly injected using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) at a flow rate of 600 μL per hour. Details of instrument tuning and calibration conditions have been previously reported (22).

Although different sample extracts were analyzed separately, the mass spectral data for each sample were combined following spectral processing. All sample peaks were calibrated using internal standards such that each internal standard mass peak had a mass error of <1 ppm relative to the theoretical mass.

4. Full-Scan Q-TOF and HPLC-Coupled Tandem Mass Spectrometry

4.1 Organic Extracts

500 μL of ethyl acetate extracts of serum from five pancreatic cancer samples and five normal samples were evaporated separately under nitrogen gas and each reconstituted in 50 μL of isopropanol:methanol:formic acid (10:89.9:0.1, v/v/v). For both LC/MS full scan and MS/MS, 20 μL of the reconstituted samples were subjected to HPLC (Agilent 1100, Agilent Technologies) analyses with Hypersil ODS column (5 μm, 150×4.6 mm), mobile phase: Solvent A: 94.9% H₂O, 5% MeOH and 0.1% Formic acid, Solvent B: 100% MeOH, gradient 100% A to 79% A and 21% B at 15 min, then to 100% B at 25 min, and then held up to 30 min at a flow rate of 1 mL/min. Eluate from the HPLC was analyzed using an ABI QSTAR® XL mass spectrometer fitted with an APCI source and data were collected in negative mode. The scan type in full scan mode was time-of-flight (TOF-MS) with a scan time of 1.0000 second, mass range between 50 and 1500 Da, and duration time of 30 min. Source parameters were as follows: Ion source gas 1 (GS1) 80; Ion source gas 2 (GS2) 10; Curtain gas (CUR) 30; Nebulizer Current (NC) −3.0; Temperature 400° C.; Declustering Potential (DP) −60; Focusing Potential (FP) −265; Declustering Potential 2 (DP2) −15. In MS/MS mode, scan type was Product Ion, scan time was 1.0000 second, scan range was 50 to 1500 Da and duration time was 30 min. All source parameters are the same as above, with collision energies (CE) of −35 V and collision gas (CAD, nitrogen) of 5.

4.2 Aqueous Extracts

10 μL of C-ACN fractions (aqueous extracts) of serum from five pancreatic cancer samples and five normal samples were directly injected into HPLC (Agilent 1100) equipped with a Meta Sil AQ column (3 μm, 100×2.0 mm, Varian) for full scan and product ion scan (MS/MS) at a flow rate of 0.18 mL/min. Solvent A: H₂O— MeOH-formic acid (94.9:5:0.1, v/v/v) and solvent B: MeOH-formic acid (99.9:0.1, v/v) were used as the mobile phase; the gradient solvent program was applied starting from 100% of A to 80% of B and 20% of A at 11 min, then held up to 20 min, then to 100% of B at 30 min, then held up to 45 min. Eluate from the HPLC was analyzed in negative and positive modes, using an Applied Biosystem (AB) QSTAR® XL mass spectrometer fitted with an ESI source. The scan type in full scan mode was time-of-flight (TOF-MS) with a scan time of 1.0000 second, mass range between 50 and 1500 Da, and duration time of 60 min. Source parameters are as follows: Ion source gas 1 (GS1), 65; Ion source gas 2 (GS2), 75; Curtain gas (CUR), 30; Temperature 425° C.; for negative mode: Ion Spray (IS), −4200V; Declustering Potential (DP), −60; Focusing Potential (FP), −265; Declustering Potential 2 (DP2), −15; and for positive mode: Ion Spray (IS), 5500V; Declustering Potential (DP), 60; Focusing Potential (FP), 265; Declustering Potential 2 (DP2), 15. In MS/MS mode, the scan type was Product Ion, scan time was set as 1.0000 second, scan range was 50 to 1500 Da and duration time was 60 min. All source parameters are the same as above, with collision energy (CE) of −30 V and +30V, respectively, and collision gas (CAD, nitrogen) of 5.

5. LC-MS/MS Flow Injection Analyses.

All LC-MS/MS analyses were performed according to Goodenowe et al. (23) with the following modifications. Specifically, analyses were performed using a triple quadrupole mass spectrometer (4000 Q TRAP, Applied Biosystems) coupled with an Agilent 1100 LC system.

5.1 MRM for C36 Markers

Sample was prepared by adding 15 L of internal standard (0.1 μg/mL of (24-¹³C)-Cholic Acid (Cambridge Isotope Laboratories, Andover, Mass.) in methanol) to 120 μL ethyl acetate fraction of each sample. 100 μL of sample was injected by flow injection analysis (FIA), and monitored under negative Atmospheric Pressure Chemical Ionization (APCI) mode. The method was based on multiple reaction monitoring (MRM) of one parent/fragment transition for each metabolite and (24-¹³C)-Cholic Acid (Table 3).

TABLE 3 List of C36 markers monitored in negative mode (organic fraction) with their formulae and transitions Predicted MRM Name Mass formula transitions “558” 558.4 C36H62O4 557.4/495.4 “574” 574.5 C36H62O5 573.5/511.4 “576” 576.5 C36H64O5 575.5/513.5 “578” 578.5 C36H66O5 577.5/515.4 “592” 592.5 C36H64O6 591.5/555.4 “594” 594.5 C36H66O6 593.5/557.5 “596” 596.5 C36H68O6 595.5/559.5

Each transition was scanned for 70 ms. 100% MeOH at a flow rate of 360 UL/min was used as the mobile phase. The source parameters were set as follows: CUR: 10.0, CAD: 8.0, NC: −4.0, TEM: 400, GS1: 30, GS2: 50, interface heater on. A standard curve was generated for all analytes to verify instrument linearity by serial dilution of (24-¹³C)-Cholic Acid in extracted commercial serum matrix (ethyl acetate fraction). All samples were analyzed in a randomized blinded manner and were bracketed by known serum standard dilutions. All standard curves had r² values>0.98.

5.2 MRM for Choline-Related Compounds

12 μL of C-ACN fraction was mixed with 108 μL mobile phase and 15 μL reserpine as an internal standard. Mobile phase consists of 75% acetonitrile and 25% of 1% formic acid in ddH₂O. 100 μL of sample was injected by flow injection analysis (FIA), and monitored under positive or negative Ion Electrospray (ESI) mode. The method was based on multiple reaction monitoring (MRM) of one parent/fragment transition for each metabolite and reserpine (Table 4). The negative ESI mode transitions for phosphatidylcholines have been selected as follows: formate adduct and qualifier (both common to same mass phosphatidylcholines), and sn-2 fatty acid (specific to individual phosphatidylcholines).

TABLE 4 List of choline-related markers with their formulae and transitions monitored in positive mode (a) and in negative mode (b), both with aqueous fractions (a) Mass MRM transitions Name (neutral) Formula (M + H) Lysophosphatidylcholines LysoPC 14:0 467.3 C22H46NO7P 468.3/184.2 LysoPC 14:1 465.3 C22H48NO7P 466.3/184.2 LysoPC 16:0 495.3 C24H50NO7P 496.3/184.2 LysoPC 16:1 493.3 C24H48NO7P 494.3/184.2 LysoPC 16:2 491.3 C24H46NO7P 492.3/184.2 LysoPC 18:0 523.3 C26H54NO7P 524.3/184.2 LysoPC 18:1 521.3 C26H52NO7P 522.3/184.2 LysoPC 18:2 519.3 C26H50NO7P 520.3/184.2 LysoPC 18:3 517.3 C26H48NO7P 518.3/184.2 LysoPC 20:1 549.4 C28H56NO7P 550.4/184.2 LysoPC 20:2 547.4 C28H54NO7P 548.4/184.2 LysoPC 20:3 545.3 C28H52NO7P 546.3/184.2 LysoPC 20:4 543.3 C28H50NO7P 544.3/184.2 LysoPC 20:5 541.3 C28H48NO7P 542.3/184.2 LysoPC 20:6 539.3 C28H46NO7P 540.3/184.2 LysoPC 22:3 573.4 C30H56NO7P 574.4/184.2 LysoPC 22:4 571.4 C30H54NO7P 572.4/184.2 LysoPC 22:5 569.4 C30H52NO7P 570.4/184.2 LysoPC 22:6 567.3 C30H50NO7P 568.3/184.2 LysoPC 24:4 599.4 C32H58NO7P 600.4/184.2 LysoPC 24:6 595.4 C32H54NO7P 596.4/184.2 LysoPC 30:1 689.5 C38H76NO7P 690.5/184.2 LysoPC 32:0 719.6 C40H82NO7P 720.6/184.2 LysoPC 32:1 717.6 C40H80NO7P 718.6/184.2 LysoPC 32:2 715.6 C40H78NO7P 716.6/184.2 LysoPC 32:6 707.5 C40H70NO7P 708.5/184.2 Phosphatidylcholines 755.6 755.55 C42H78NO8P 756.6/184.2 757.6 757.56 C42H80N08P 758.6/184.2 759.6 759.58 C42H82NO8P 760.6/184.2 761.6 761.59 C42H84NO8P 762.6/184.2 781.6 781.56 C44H80NO8P 782.6/184.2 783.6 783.58 C44H82NO8P 784.6/184.2 785.6 785.59 C44H84NO8P 786.6/184.2 787.6 787.61 C44H86NO8P 788.6/184.2 805.6 805.56 C46H80NO8P 806.6/184.2 807.6 807.58 C46H82NO8P 808.6/184.2 809.6 809.59 C46H84NO8P 810.6/184.2 831.6 831.58 C48H82NO8P 832.6/184.2 833.6 833.59 C48H84NO8P 834.6/184.2 Plasmenylcholines 742.6 741.57 C42H80NO7P 742.6/184.2 744.6 743.58 C42H82NO7P 744.6/184.2 746.6 745.60 C42H84NO7P 746.6/184.2 768.6 767.58 C44H82NO7P 768.6/184.2 770.6 769.60 C44H84NO7P 770.6/184.2 772.6 771.61 C44H86NO7P 772.6/184.2 774.6 773.63 C44H88NO7P 774.6/184.2 792.6 791.58 C46H82NO7P 792.6/184.2 794.6 793.60 C46H84NO7P 794.6/184.2 796.6 795.61 C46H86NO7P 796.6/184.2 818.6 817.60 C48H84NO7P 818.6/184.2 820.6 819.61 C48H86NO7P 820.6/184.2 Molecular Mass Metabolite Name Formula (neutral) MRM Transition Sphingomyelins SM(d18:1/16:0) C₃₉H₇₉N₂O₆P 702.5 703.6/184.2 SM(d18:1/18:1) C₄₁H₈₁N₂O₆P 728.5 729.6/184.2 SM(d18:1/18:0) C₄₁H₈₃N₂O₆P 730.5 731.6/184.2 SM(d18:1/24:1 (15Z)) C₄₇H₉₃N₂O₆P 812.5 813.6/184.2 SM(d18:1/24:0) C₄₇H₉₅N₂O₆P 814.5 815.6/184.2 (b) Parent Molecular Mass [M + FA − H] Metabolite Name Formula (neutral] Mass MRM Transitions Lysophosphatidylcholines LysoPC 14:0 C22H46NO7P 467.3 512.3 512.3/452.3/227.2 LysoPC 14:1 C22H44NO7P 465.3 510.3 510.3/450.3/225.2 LysoPC 16:0 C24H50NO7P 495.3 540.3 540.3/480.3/255.2 LysoPC 16:1 C24H48NO7P 493.3 538.3 538.3/478.3/253.2 LysoPC 16:2 C24H46NO7P 491.3 535.3 536.3/476.3/251.2 LysoPC 18:0 C26H54NO7P 523.4 568.4 568.4/508.4/283.3 LysoPC 18:1 C26H52NO7P 521.3 566.3 566.3/506.3/281.3 LysoPC 18:2 C26H50NO7P 519.3 564.3 564.3/504.3/279.3 LysoPC 18:3 C26H48NO7P 517.3 562.3 562.3/502.3/277.3 LysoPC 20:1 C28H56NO7P 549.4 594.4 594.4/534.4/309.3 LysoPC 20:2 C28H54NO7P 547.4 592.4 592.4/532.4/307.3 LysoPC 20:3 C28H52NO7P 545.3 590.3 590.3/530.3/305.2 LysoPC 20:4 C28H50NO7P 543.3 588.3 588.3/528.3/303.2 LysoPC 20:5 C28H48NO7P 541.3 586.3 586.3/526.3/301.2 LysoPC 20:6 C28H46NO7P 539.3 584.3 584.3/524.3/299.2 LysoPC 22:3 C30H56NO7P 573.4 618.4 618.4/558.4/333.3 LysoPC 22:4 C30H54NO7P 571.4 616.4 616.4/556.4/331.3 LysoPC 22:5 C30H52NO7P 559.3 614.3 614.3/554.3/329.2 LysoPC 22:6 C30H50NO7P 567.3 612.3 612.3/552.3/327.2 LysoPC 24:4 C32H58NO7P 599.4 644.4 644.4/584.4/359.3 LysoPC 24:6 C32H54NO7P 595.4 640.4 640.4/580.4/355.3 LysoPC 30:1 C38H76NO7P 589.5 734.5 734.5/674.5/449.4 LysoPC 32:0 C40H82NO7P 719.6 764.6 764.6/703.6/479.5 LysoPC 32:1 C40H80NO7P 717.6 762.6 762.6/702.6/477.4 LysoPC 32:2 C40H78NO7P 715.6 760.6 760.6/700.6/475.4 LysoPC 32:6 C40H70NO7P 707.5 752.5 752.5/692.5/467.4 Parent Molecular Metabolite Name Mass Formula [Parent + FA − H] MRM Transitions Phosphatidylcholines PtdCho 16:0/18:3 755.6 C42H78NO8P 800.6 800.6/740.6/277.2 PtdCho 16:1/18:2 755.6 C42H78NO8P 800.6 800.6/740.6/279.2 PtdCho 18:2/16:1 755.6 C42H78NO8P 800.6 800.6/740.6/253.2 PtdCho 18:3/16:0 755.6 C42H78NO8P 800.6 800.6/740.6/255.2 PtdCho 16:0/18:2 757.6 C42H80NO8P 802.6 802.6/742.6/279.2 PtdCho 16:1/18:1 757.6 C42H80NO8P 802.6 802.6/742.6/281.2 PtdCho 18:1/16:1 757.6 C42H80NO8P 802.6 802.6/742.6/253.2 PtdCho 18:2/16:0 757.6 C42H80NO8P 802.6 802.6/742.6/255.2 PtdCho 16:0/18:1 759.6 C42H82NO8P 804.6 804.6/744.6/281.2 PtdCho 18:1/16:0 759.6 C42H82NO8P 804.6 804.6/744.6/255.2 PtdCho 18:0/16:0 761.6 C42H84NO8P 806.6 806.6/746.6/255.2 PtdCho 16:0/18:0 761.6 C42H84NO8P 806.6 806.6/746.6/281.2 PtdCho 16:0/20:5 779.6 C44H78NO8P 824.6 824.6/764.6/301.2 PtdCho 18:3/18:2 779.6 C44H78NO8P 824.6 824.6/764.6/279.2 PtdCho 20:5/16:0 779.6 C44H78NO8P 824.6 824.6/764.6/255.2 PtdCho 16:0/20:4 781.6 C44H80NO8P 826.6 826.6/766.6/303.2 PtdCho 18:2/18:2 781.6 C44H80NO8P 826.6 826.6/766.6/279.2 PtdCho 20:4/16:0 781.6 C44H80NO8P 826.6 826.6/766.6/255.2 PtdCho 16:0/20:3 783.6 C44H82NO8P 828.6 828.6/768.6/305.3 PtdCho 18:1/18:2 783.6 C44H82NO8P 828.6 828.6/768.6/279.2 PtdCho 18:2/18:1 783.6 C44H82NO8P 828.6 828.6/768.6/281.2 PtdCho 20:3/16:0 783.6 C44H82NO8P 828.6 828.6/768.6/255.2 PtdCho 18:0/18:2 785.6 C44H84NO8P 830.6 830.6/770.6/279.2 PtdCho 18:1/18:1 785.6 C44H84NO8P 830.6 830.6/770.6/281.2 PtdCho 18:2/18:0 785.6 C44H84NO8P 830.6 830.6/770.6/283.2 PtdCho 18:0/18:1 787.6 C44H86NO8P 832.6 832.5/772.6/281.2 PtdCho 18:1/18:0 787.6 C44H86NO8P 832.6 832.5/772.6/283.2 PtdCho 18:0/18:0 789.6 C44H88NO8P 834.6 834.6/774.6/283.2 PtdCho 16:1/22:6 803.6 C46H78NO8P 848.6 848.6/788.6/327.3 PtdCho 20:5/18:2 803.6 C46H78NO8P 848.6 848.6/788.6/279.2 PtdCho 16:0/22:6 805.6 C46H80NO8P 850.6 850.6/790.6/327.3 PtdCho 18:2/20:4 805.6 C46H80NO8P 850.6 850.6/790.6/303.2 PtdCho 20:4/18:2 805.6 C46H80NO8P 850.6 850.6/790.6/279.2 PtdCho 22:6/16:0 805.6 C46H80NO8P 850.6 850.6/790.6/255.2 PtdCho 16:0/22:5 807.6 C46H82NO8P 852.6 852.6/792.6/329.3 PtdCho 18:0/20:5 807.6 C46H82NO8P 852.6 852.6/792.6/301.3 PtdCho 18:1/20:4 807.6 C46H82NO8P 852.6 852.6/792.6/303.2 PtdCho 20:4/18:1 807.6 C46H82NO8P 852.6 852.6/792.6/281.2 PtdCho 20:5/18:0 807.6 C46H82NO8P 852.6 852.6/792.6/283.2 PtdCho 22:5/16:0 807.6 C46H82NO8P 852.6 852.6/792.6/255.2 PtdCho 16:0/22:4 809.6 C46H84NO8P 854.6 854.6/794.6/331.3 PtdCho 18:0/20:4 809.6 C46H84NO8P 854.6 854.6/794.6/303.2 PtdCho 20:4/18:0 809.6 C46H84NO8P 854.6 854.6/794.6/283.2 PtdCho 22:4/16:0 809.6 C46H84NO8P 854.6 854.6/794.6/255.2 PtdCho 18:1/22:6 831.6 C48H82NO8P 876.6 876.6/916.6/327.3 PtdCho 22:6/18:1 831.6 C48H82NO8P 876.6 876.6/916.6/281.2 PtdCho 18:0/22:6 833.6 C48H84NO8P 878.6 878.6/818.6/327.3 PtdCho 22:6/18:0 833.6 C48H84NO8P 878.6 878.6/818.6/283.2 PtdCho 18:0/22:5 835.6 C48H86NO8P 880.6 880.6/820.6/329.3 PtdCho 22:5/18:0 835.6 C48H86NO8P 880.6 880.6/820.6/283.2 Molecular Parent [M + FA − H] Metabolite Name Formula Mass Mass MRM Transitions Sphingomyelins SM(d18:1/16:0) C₃₉H₇₉N₂O₆P 702.6 747.6 747.6/687.6/168.1 SM(d18:1/18:1) C₄₁H₈₁N₂O₆P 728.6 773.6 773.6/713.6/168.1 SM(d18:1/18:0) C₄₁H₈₃N₂O₆P 730.6 775.6 775.6/715.6/168.1 SM(d18:1/24:1 (15Z)) C₄₇H₉₃N₂O₆P 812.6 857.6 857.6/797.6/168.1 SM(d18:1/24:0) C₄₇H₉₅N₂O₆P 814.6 859.6 859.6/799.6/168.1

Each transition was scanned for 70 ms. Mobile phase was used at a flow rate of 60 μL/min. The source parameters were set as follows: CUR: 10.0, IS: 5500.0, CAD: 10.0, TEM: 500, GS1: 30, GS2: 50, interface heater on. A standard curve was generated for all analytes to verify instrument linearity by serial dilution of C-ACN fraction of Randox (Human Serum Precision Control Level II) with constant concentration of reserpine. All samples were analyzed in a randomized blinded manner and were bracketed by known serum standard dilutions. All standard curves had r² values>0.98. For sphingomyelins, both MRM transitions were run and similarity was verified; the MRM transitions with m/z 168 were selected for the graphs reported.

6. Statistical Analysis

FTICR-MS accurate mass array alignments were performed using DISCOVAmetrics™ (Phenomenome Discoveries Inc., Saskatoon). Initial statistical analysis and graphs of FTICR-MS data were carried out using Microsoft Office Excel 2007. Two-tailed unpaired Student's t-tests were used for determination of significant difference between pancreatic cancer and controls. P-values of less than 0.05 were considered significant. ROC curves were generated from logistic regression analysis using SAS Enterprise Guide 4.2.

Results FTICR Metabolomic Profiling

1A. FTICR Data Analysis

The experimental workflow generated for the studies described here is summarized in FIG. 1.

Serum metabolites were captured through a liquid extraction process (see methods) and extracts were directly infused by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) on an FTICR mass spectrometer. In total six separate analyses comprising combinations of extracts and ionization modes were obtained for each sample:

-   -   Aqueous Extract         -   1. Positive ESI (analysis mode 1101)         -   2. Negative ESI (analysis mode 1102)     -   Organic Extract         -   3. Positive ESI (analysis mode 1201)         -   4. Negative ESI (analysis mode 1202)         -   5. Positive APCI (analysis mode 1203)         -   6. Negative APCI (analysis mode 1204)

Separately for each project, the resulting spectral data of all the subjects was aligned within 1 ppm mass accuracy, background peaks were subtracted, and a two-dimensional array table comprising the intensities of each of the sample-specific spectral peaks was created using custom informatics software DISCOVAmetrics™.

In the metabolomic profile thus created, a Boolean filtering sorted the masses that differentiate the “pancreatic cancer” condition from the “control” condition. Table 5 lists the 362 masses that discriminate the pancreatic cancer samples from the control samples with a p-value lower than 0.05.

TABLE 5 Accurate mass features differing between clinically diagnosed pancreatic cancer patients and controls (p < 0.05). Ratio pancreatic AVG Detected Analysis cancer/ AVG pancreatic Mass Mode P value control controls cancer 786.593 1101 5.24E−14 0.30 15.59 4.61 595.4897 1202 7.48E−14 0.36 5.30 1.88 594.4863 1202 9.91E−14 0.31 14.02 4.40 785.5913 1101 1.39E−13 0.27 33.03 8.93 808.5783 1101 1.63E−13 0.30 15.62 4.75 702.5709 1101 2.39E−13 0.47 9.60 4.48 780.5452 1101 3.57E−13 0.30 22.48 6.68 807.5734 1101 5.49E−13 0.28 34.47 9.70 576.4751 1202 5.61E−13 0.40 4.85 1.93 541.3134 1101 6.66E−13 0.37 6.27 2.30 804.5422 1101 2.04E−12 0.34 6.56 2.26 779.5405 1101 2.66E−12 0.26 53.60 13.93 812.6774 1101 3.81E−12 0.54 5.11 2.77 758.5626 1101 1.17E−11 0.31 23.80 7.28 783.569 1101 1.19E−11 0.36 13.94 4.98 596.5017 1202 2.03E−11 0.36 11.29 4.04 803.5373 1101 2.11E−11 0.33 13.46 4.48 810.5867 1101 3.75E−11 0.39 7.46 2.90 724.5477 1101 3.75E−11 0.49 8.07 3.98 519.3295 1101 7.30E−11 0.41 6.62 2.71 757.556 1101 1.04E−10 0.27 58.48 16.01 600.5117 1203 2.61E−10 1.40 124.61 174.25 809.5796 1101 2.67E−10 0.43 15.81 6.73 829.5516 1101 2.82E−10 0.41 7.32 2.98 523.3661 1101 3.97E−10 0.49 4.95 2.44 784.5742 1101 4.29E−10 0.42 6.06 2.54 806.5632 1101 4.47E−10 0.37 13.07 4.77 601.5151 1203 5.26E−10 1.39 52.15 72.62 805.5549 1101 6.17E−10 0.35 27.28 9.66 833.5864 1101 9.05E−10 0.43 9.23 4.01 723.5203 1202 1.35E−09 0.55 6.92 3.80 749.5374 1202 1.36E−09 0.46 11.63 5.39 782.5612 1101 1.71E−09 0.37 19.17 7.08 827.5401 1101 1.73E−09 0.39 12.52 4.83 801.5147 1101 2.21E−09 0.39 6.00 2.34 834.5868 1101 2.61E−09 0.45 4.76 2.16 781.5566 1101 4.33E−09 0.33 44.71 14.95 828.5397 1101 4.68E−09 0.41 6.34 2.61 831.5652 1101 4.96E−09 0.51 8.53 4.33 592.4709 1202 5.85E−09 0.37 4.97 1.85 759.5383 1101 9.35E−09 0.53 11.72 6.21 240.0997 1202 1.36E−08 0.45 15.83 7.05 1038.915 1203 1.58E−08 0.39 6.28 2.45 588.3269 1202 1.79E−08 0.54 6.01 3.25 587.3214 1202 2.93E−08 0.50 19.54 9.71 545.3454 1101 4.01E−08 0.54 4.67 2.53 382.1601 1201 4.69E−08 1.94 12.90 24.98 326.2048 1202 5.08E−08 2.58 3.05 7.87 360.1782 1201 7.10E−08 1.52 5.83 8.85 280.2404 1202 7.61E−08 2.44 16.22 39.65 281.2432 1202 9.00E−08 2.38 3.68 8.77 214.1204 1203 1.01E−07 1.67 6.67 11.12 302.222 1201 1.19E−07 2.58 13.20 34.07 282.2558 1202 1.47E−07 2.40 31.82 76.36 575.4985 1203 1.73E−07 1.25 61.51 76.85 855.5721 1101 1.74E−07 0.39 4.56 1.77 283.2591 1202 1.89E−07 2.45 6.15 15.06 759.5733 1101 2.33E−07 0.39 31.65 12.32 760.5792 1101 2.65E−07 0.45 13.99 6.28 574.4952 1203 2.84E−07 1.25 162.04 201.84 517.3141 1101 4.79E−07 0.57 16.74 9.61 283.2595 1204 5.17E−07 1.41 11.22 15.84 262.0814 1201 5.40E−07 0.44 10.65 4.71 811.5729 1202 5.45E−07 0.65 6.67 4.34 1040.933 1203 6.01E−07 0.58 10.18 5.93 328.2627 1202 6.71E−07 2.06 16.89 34.73 326.2458 1202 7.57E−07 2.06 7.72 15.91 282.2559 1204 8.36E−07 1.46 56.00 81.98 564.5121 1202 9.99E−07 3.32 3.17 10.53 276.0948 1201 1.00E−06 1.22 9.93 12.12 775.5522 1202 1.02E−06 0.51 7.83 4.02 811.608 1101 1.38E−06 0.49 4.23 2.09 824.69 1203 1.74E−06 0.61 5.01 3.03 495.3325 1101 2.06E−06 0.58 17.63 10.17 508.2256 1201 2.26E−06 1.43 4.64 6.65 562.4962 1202 2.46E−06 2.91 3.06 8.90 329.2658 1202 2.48E−06 1.99 3.85 7.68 518.321 1101 2.57E−06 0.63 4.70 2.95 1016.931 1203 3.03E−06 0.57 58.00 32.85 1017.935 1203 3.05E−06 0.57 45.70 26.16 360.1792 1202 5.30E−06 1.49 36.91 54.94 566.3403 1202 5.63E−06 0.67 29.61 19.78 565.3373 1202 5.77E−06 0.65 118.24 77.43 300.2067 1201 6.14E−06 2.41 2.57 6.20 771.5699 1202 7.10E−06 0.69 6.90 4.76 116.5696 1202 7.19E−06 1.22 5.54 6.78 468.3807 1202 8.42E−06 0.64 5.22 3.33 361.1828 1202 8.72E−06 1.50 7.05 10.58 428.3647 1201 9.73E−06 0.66 10.24 6.78 1255.153 1203 1.04E−05 0.59 7.38 4.36 1200.088 1203 1.52E−05 0.55 8.45 4.64 540.4381 1202 1.58E−05 0.61 5.38 3.27 851.7107 1203 1.63E−05 0.72 8.61 6.17 1018.944 1203 1.71E−05 0.64 38.50 24.48 505.3146 1202 1.95E−05 0.73 6.13 4.50 496.3373 1101 2.20E−05 0.65 4.79 3.10 569.3682 1202 2.30E−05 0.72 39.22 28.31 330.2559 1202 2.43E−05 2.07 4.21 8.70 808.5791 1201 2.54E−05 0.71 46.64 33.33 572.4798 1203 3.15E−05 1.20 18.65 22.42 765.5678 1201 3.27E−05 0.77 5.30 4.06 786.5972 1201 3.39E−05 0.72 29.54 21.13 1228.117 1203 3.63E−05 0.64 24.71 15.87 791.5841 1201 4.14E−05 0.75 5.81 4.35 1229.12 1203 4.19E−05 0.61 13.11 7.98 850.7061 1203 4.30E−05 0.72 13.39 9.66 830.5591 1201 4.47E−05 0.70 13.41 9.34 1201.09 1203 4.63E−05 0.50 9.82 4.92 802.5291 1201 4.75E−05 0.60 12.32 7.38 1041.935 1203 5.64E−05 0.65 7.35 4.79 260.0033 1101 6.07E−05 1.35 7.68 10.34 785.5929 1201 6.59E−05 0.71 70.58 50.30 1227.112 1203 6.68E−05 0.65 32.33 20.98 826.5561 1202 7.07E−05 0.50 15.42 7.74 1199.084 1203 7.39E−05 0.63 10.34 6.53 825.5522 1202 8.54E−05 0.47 32.08 15.06 244.0554 1101 8.97E−05 1.36 8.98 12.19 602.5269 1203 9.14E−05 1.26 208.79 262.81 570.372 1202 9.17E−05 0.76 11.10 8.43 599.4993 1203 9.78E−05 1.25 15.04 18.83 1019.951 1203 9.91E−05 0.67 21.64 14.54 1039.705 1201 1.01E−04 0.73 4.53 3.29 573.4833 1203 1.03E−04 1.19 7.23 8.57 801.5262 1201 1.06E−04 0.55 30.09 16.68 603.5297 1203 1.09E−04 1.25 86.64 108.65 1230.125 1203 1.10E−04 0.50 5.34 2.69 317.9613 1101 1.28E−04 1.39 5.16 7.18 807.5739 1201 1.34E−04 0.74 115.80 86.22 598.4955 1203 1.47E−04 1.25 37.42 46.65 368.1057 1202 1.61E−04 1.35 4.89 6.61 280.2403 1204 1.62E−04 1.24 31.44 39.14 823.5411 1201 1.65E−04 0.77 5.10 3.95 1039.921 1203 1.68E−04 0.52 4.79 2.48 284.9259 1203 1.69E−04 1.26 6.30 7.96 270.0867 1201 1.72E−04 1.19 20.78 24.82 578.5169 1203 1.75E−04 1.33 21.27 28.33 948.836 1204 1.83E−04 0.67 10.17 6.85 446.3395 1202 1.85E−04 0.70 5.27 3.69 577.5149 1203 1.90E−04 1.23 119.24 147.02 633.3245 1202 2.02E−04 0.68 8.26 5.63 590.3408 1202 2.15E−04 0.75 11.13 8.39 837.7209 1204 2.38E−04 0.61 6.81 4.19 469.3616 1201 2.44E−04 0.72 5.54 3.97 468.3581 1201 2.46E−04 0.69 17.82 12.36 856.7505 1203 2.49E−04 1.22 205.07 250.07 576.5113 1203 2.52E−04 1.23 316.44 388.31 522.4639 1203 2.60E−04 0.62 16.82 10.39 787.5989 1101 2.64E−04 0.63 9.66 6.13 589.3368 1202 2.93E−04 0.74 35.28 26.06 300.1186 1202 3.03E−04 1.28 11.97 15.32 831.5997 1202 3.11E−04 0.66 72.40 47.60 270.0323 1101 3.20E−04 1.34 13.66 18.30 281.2435 1204 3.34E−04 1.23 6.03 7.44 84.0575 1202 3.34E−04 1.22 6.64 8.13 856.754 1204 3.41E−04 1.22 44.96 54.67 922.8222 1204 3.47E−04 0.53 8.16 4.32 832.6031 1202 3.48E−04 0.67 34.86 23.36 1202.098 1203 3.56E−04 0.58 7.40 4.27 829.5532 1201 3.74E−04 0.69 34.32 23.60 857.7543 1203 3.97E−04 1.21 114.80 138.68 327.9902 1101 4.25E−04 1.36 5.92 8.05 304.2407 1202 4.27E−04 1.46 8.00 11.67 538.4237 1202 4.40E−04 0.63 6.33 3.97 1020.957 1203 4.47E−04 0.69 7.99 5.52 1250.108 1203 4.49E−04 0.56 5.60 3.16 1253.134 1203 4.55E−04 0.63 11.69 7.34 847.531 1201 4.82E−04 0.78 5.86 4.56 200.1389 1202 5.57E−04 1.34 6.87 9.21 350.2222 1201 5.59E−04 1.74 4.00 6.97 857.7574 1204 5.87E−04 1.20 25.77 30.92 203.1155 1101 6.19E−04 1.49 7.01 10.46 197.0896 1101 7.68E−04 1.34 5.71 7.68 523.4675 1203 8.74E−04 0.64 5.97 3.84 191.5055 1203 9.26E−04 1.31 9.55 12.54 1011.669 1201 9.48E−04 0.78 6.76 5.27 838.7284 1204 9.60E−04 0.62 4.89 3.04 338.0189 1101 9.69E−04 1.34 7.96 10.70 202.045 1101 1.04E−03 1.32 33.71 44.61 302.0945 1201 1.06E−03 1.26 10.37 13.02 873.7819 1203 1.08E−03 1.23 8.50 10.45 1225.096 1203 1.15E−03 0.71 25.00 17.85 446.2526 1204 1.15E−03 2.33 2.87 6.69 898.7043 1203 1.31E−03 0.56 3.34 1.86 382.1083 1101 1.33E−03 1.56 5.59 8.70 970.733 1204 1.38E−03 0.55 6.35 3.49 715.6959 1101 1.42E−03 2.04 5.53 11.27 302.2457 1202 1.45E−03 1.23 9.18 11.33 851.7337 1204 1.56E−03 0.65 5.80 3.78 874.787 1203 1.64E−03 1.29 4.60 5.92 721.5035 1204 1.69E−03 0.48 3.57 1.70 630.799 1101 1.70E−03 2.32 25.23 58.48 1252.12 1203 1.70E−03 0.64 7.72 4.94 268.1284 1201 1.77E−03 1.34 8.63 11.54 780.5454 1201 1.80E−03 0.77 71.95 55.17 750.5425 1204 1.91E−03 0.46 8.04 3.67 749.5388 1204 1.96E−03 0.43 17.32 7.50 947.8263 1204 1.97E−03 0.77 15.18 11.72 853.573 1202 2.04E−03 0.67 26.48 17.62 779.5416 1201 2.06E−03 0.80 169.63 135.27 1224.096 1203 2.07E−03 0.70 9.01 6.26 838.7435 1203 2.13E−03 1.21 8.28 10.01 1226.599 1203 2.20E−03 0.73 20.59 15.07 635.7525 1101 2.21E−03 2.25 34.61 77.78 871.5547 1202 2.24E−03 0.80 8.04 6.45 743.5396 1202 2.25E−03 0.80 14.69 11.72 924.7233 1203 2.30E−03 0.61 9.77 5.92 801.5523 1202 2.44E−03 0.72 7.08 5.07 615.3535 1202 2.48E−03 0.77 7.10 5.50 541.3361 1202 2.58E−03 0.79 104.55 82.62 921.813 1204 2.60E−03 0.75 19.33 14.41 520.448 1203 2.72E−03 0.69 6.51 4.49 903.7636 1204 2.80E−03 1.19 105.15 125.04 744.5425 1202 2.99E−03 0.78 6.66 5.18 318.0931 1202 3.14E−03 0.82 20.17 16.54 758.562 1201 3.16E−03 0.77 64.96 49.94 1254.137 1203 3.19E−03 0.71 8.83 6.24 868.7704 1204 3.38E−03 0.68 3.94 2.67 606.5591 1203 3.47E−03 0.44 4.80 2.11 998.7566 1204 3.50E−03 0.74 10.82 7.99 329.2439 1202 3.53E−03 1.46 7.29 10.65 594.4852 1204 3.63E−03 0.59 11.81 7.00 757.5587 1201 3.64E−03 0.80 161.90 129.94 925.727 1203 3.69E−03 0.58 6.16 3.57 996.7518 1204 3.73E−03 0.67 11.29 7.51 804.5714 1202 3.76E−03 0.74 81.05 59.96 595.4892 1204 3.81E−03 0.61 4.70 2.86 328.2408 1202 3.92E−03 1.46 28.17 41.15 1223.09 1203 4.15E−03 0.73 9.81 7.16 803.5677 1202 4.22E−03 0.74 169.16 125.07 752.5574 1204 4.28E−03 0.54 7.20 3.87 328.2403 1204 4.36E−03 1.40 5.10 7.15 332.1473 1202 4.52E−03 1.21 7.74 9.34 631.798 1101 4.72E−03 1.92 3.52 6.76 775.5532 1204 5.06E−03 0.46 14.20 6.58 777.5709 1204 5.40E−03 0.54 6.39 3.44 636.7532 1101 5.40E−03 2.05 4.43 9.09 867.7649 1204 5.52E−03 0.71 7.81 5.51 597.5066 1204 5.52E−03 0.62 4.55 2.81 908.7907 1204 5.56E−03 0.68 9.63 6.54 763.5578 1204 5.62E−03 0.57 3.17 1.79 596.5027 1204 5.84E−03 0.60 11.58 6.97 777.0402 1204 6.01E−03 0.52 6.89 3.59 542.3394 1202 6.53E−03 0.83 23.67 19.76 723.521 1204 6.76E−03 0.57 7.41 4.19 627.5656 1203 6.89E−03 1.26 5.47 6.87 657.7337 1101 6.92E−03 2.06 20.13 41.54 255.1161 1201 7.01E−03 1.14 27.21 30.97 751.5511 1202 7.02E−03 0.64 7.10 4.57 751.5539 1204 7.02E−03 0.53 15.18 8.11 827.5678 1202 7.35E−03 0.71 67.73 47.91 658.7372 1101 7.35E−03 1.91 2.70 5.15 804.5456 1201 7.48E−03 0.79 26.05 20.68 670.5696 1203 7.50E−03 0.68 10.09 6.81 628.5438 1203 7.58E−03 1.18 7.10 8.39 613.3379 1202 7.62E−03 0.81 36.81 29.89 645.7958 1101 7.76E−03 2.00 3.94 7.88 850.7326 1204 7.89E−03 0.70 6.57 4.60 923.7295 1204 7.93E−03 0.83 13.51 11.27 579.5313 1203 8.30E−03 0.70 12.95 9.10 748.527 1204 8.77E−03 0.52 5.95 3.07 783.5755 1201 9.29E−03 0.79 37.00 29.41 828.5721 1202 9.38E−03 0.73 31.90 23.31 578.5284 1203 9.41E−03 0.71 33.06 23.56 894.7911 1204 9.58E−03 0.77 18.12 14.02 910.7272 1204 9.85E−03 0.83 10.17 8.45 112.0974 1201 1.01E−02 1.19 7.97 9.46 857.6923 1204 1.02E−02 0.49 2.58 1.26 1012.781 1204 1.03E−02 0.71 7.04 4.99 733.5054 1204 1.06E−02 1.35 6.61 8.91 829.5843 1202 1.08E−02 0.75 38.25 28.65 855.7436 1204 1.09E−02 1.15 12.81 14.70 997.7397 1204 1.09E−02 0.69 10.03 6.88 984.7406 1204 1.13E−02 0.73 7.01 5.09 735.6582 1204 1.13E−02 0.74 7.76 5.74 830.5879 1202 1.18E−02 0.77 18.18 13.95 775.5532 1203 1.19E−02 0.57 2.87 1.64 902.7629 1204 1.28E−02 1.16 113.63 131.44 874.7066 1203 1.29E−02 0.76 8.79 6.67 861.749 1203 1.30E−02 0.79 7.93 6.25 243.0714 1101 1.32E−02 1.24 7.52 9.33 256.2403 1202 1.33E−02 1.21 10.40 12.63 766.4792 1204 1.34E−02 0.70 5.88 4.13 214.1205 1201 1.34E−02 1.15 22.10 25.34 854.7397 1204 1.41E−02 1.15 19.42 22.33 1249.105 1203 1.45E−02 0.72 6.51 4.67 795.5181 1201 1.46E−02 0.84 11.33 9.57 854.7358 1203 1.48E−02 1.17 164.45 192.27 946.8194 1204 1.55E−02 0.81 26.05 21.20 719.6256 1204 1.56E−02 1.30 8.46 10.99 919.6496 1101 1.56E−02 1.57 1.25 1.96 1251.119 1203 1.58E−02 0.72 9.37 6.76 855.7392 1203 1.60E−02 1.17 95.69 111.76 671.5731 1203 1.67E−02 0.72 5.22 3.74 839.7464 1203 1.71E−02 1.19 5.07 6.01 933.8137 1204 1.72E−02 0.80 21.43 17.14 725.7228 1101 1.74E−02 1.76 4.71 8.28 916.7735 1204 1.78E−02 1.15 137.59 158.45 468.2336 1201 1.80E−02 1.36 22.33 30.32 804.7208 1203 1.91E−02 0.70 5.47 3.81 304.2375 1201 1.92E−02 1.71 7.28 12.43 922.7285 1204 1.92E−02 0.81 15.80 12.85 609.3259 1202 1.93E−02 0.83 8.39 6.98 755.5497 1201 1.98E−02 0.84 5.36 4.49 972.7481 1204 2.01E−02 0.79 9.99 7.91 827.7082 1203 2.03E−02 0.85 9.17 7.79 494.4321 1203 2.04E−02 0.59 3.30 1.96 232.1309 1202 2.05E−02 1.09 227.50 248.81 803.5414 1201 2.06E−02 0.81 66.42 53.85 826.7047 1203 2.17E−02 0.85 15.48 13.19 720.6272 1204 2.20E−02 1.27 4.46 5.67 807.5764 1203 2.20E−02 0.71 3.47 2.46 922.7081 1203 2.29E−02 0.62 2.64 1.62 986.7568 1204 2.29E−02 0.83 9.18 7.65 348.1191 1201 2.29E−02 0.79 5.78 4.58 813.5888 1202 2.33E−02 0.84 5.27 4.43 233.1345 1202 2.41E−02 1.10 27.50 30.33 784.5806 1201 2.48E−02 0.85 14.21 12.08 973.7482 1204 2.50E−02 0.83 9.22 7.69 724.5252 1204 2.56E−02 0.69 3.95 2.71 1011.77 1204 2.62E−02 0.72 6.37 4.59 858.7644 1203 2.64E−02 1.15 121.34 139.48 835.598 1201 2.84E−02 0.86 6.87 5.90 469.237 1201 2.88E−02 1.28 5.11 6.54 773.5276 1204 2.94E−02 0.74 12.51 9.32 889.7537 1204 2.97E−02 1.13 79.81 90.07 819.5177 1201 3.10E−02 0.86 5.89 5.09 875.7108 1203 3.11E−02 0.78 5.01 3.89 781.5029 1204 3.18E−02 0.75 6.97 5.26 793.7091 1101 3.19E−02 1.68 4.56 7.67 866.7585 1204 3.28E−02 0.79 17.66 13.95 785.5931 1203 3.30E−02 0.78 5.98 4.67 485.904 1101 3.46E−02 1.14 7.86 8.96 1253.123 1201 3.47E−02 0.69 3.56 2.45 481.315 1202 3.56E−02 0.90 9.09 8.20 745.5631 1203 3.64E−02 1.47 7.05 10.35 851.6694 1101 3.64E−02 1.59 1.78 2.84 1010.765 1204 3.71E−02 0.72 8.26 5.97 999.7632 1204 3.72E−02 0.81 8.01 6.52 907.7847 1204 3.78E−02 0.81 23.16 18.73 254.1127 1201 3.80E−02 1.13 215.52 243.63 898.7325 1204 3.80E−02 0.88 13.60 11.95 418.2204 1204 4.01E−02 0.61 12.12 7.44 522.4638 1201 4.01E−02 0.67 3.86 2.59 937.7542 1204 4.06E−02 0.88 18.10 15.92 484.3527 1201 4.09E−02 0.74 11.34 8.43 366.3593 1101 4.15E−02 1.81 2.10 3.80 852.7368 1204 4.16E−02 0.88 7.42 6.52 831.572 1201 4.16E−02 0.84 30.16 25.20 746.5128 1204 4.27E−02 1.27 10.08 12.78 796.5212 1201 4.29E−02 0.85 4.71 3.98 1247.084 1203 4.37E−02 0.71 3.97 2.83 889.8147 1203 4.41E−02 0.65 1.97 1.28 681.5858 1204 4.42E−02 0.78 4.60 3.60 746.5705 1204 4.44E−02 1.31 7.89 10.30 865.752 1204 4.49E−02 0.81 28.03 22.72 960.7432 1204 4.59E−02 0.87 10.60 9.21 950.7364 1203 4.73E−02 0.72 14.31 10.32 78.0516 1202 4.75E−02 1.09 4.89 5.32 774.5419 1204 4.76E−02 0.72 6.38 4.61 428.2404 1201 4.93E−02 1.35 3.83 5.15 879.7629 1204 4.97E−02 0.79 24.59 19.43 909.7882 1203 4.98E−02 1.12 18.12 20.25

Principal Component Analysis was then performed on the whole populations (90 samples) upon the 362 markers through DISCOVAmetrics™. FIG. 2 illustrates the separation resulting from this unsupervised classification between pancreatic cancer (with individual samples in grey) and controls (in black).

¹³C isotopic peaks were identified, such as the first two markers, 786.593 and 595.4897, which are the isotopic peaks of the fourth and third markers respectively, 785.5913 and 594.4863. Table 6 lists the 20 best biomarkers without ¹³C isotopic peaks. All of these markers except 600.5117 have decreased levels in the pancreatic cancer cohort relative to controls.

TABLE 6 List of the 20 best FTICR biomarkers of pancreatic cancer, sorted by mass within their analysis mode. Ratio Analysis Detected pancreatic Mode Mass P value cancer/control 1101 519.3295 7.30E−11 0.41 523.3661 3.97E−10 0.49 541.3134 6.66E−13 0.37 702.5709 2.39E−13 0.47 724.5477 3.75E−11 0.49 757.556 1.04E−10 0.27 779.5405 2.66E−12 0.26 783.569 1.19E−11 0.36 785.5913 1.39E−13 0.27 803.5373 2.11E−11 0.33 805.5549 6.17E−10 0.35 807.5734 5.49E−13 0.28 809.5796 2.67E−10 0.43 812.6774 3.81E−12 0.54 829.5516 2.82E−10 0.41 833.5864 9.05E−10 0.43 1202 576.4751 5.61E−13 0.40 594.4863 9.91E−14 0.31 596.5017 2.03E−11 0.36 1203 600.5117 2.61E−10 1.40

Principal Component Analysis was then performed on the whole populations upon these 20 markers through DISCOVAmetrics™. FIG. 3 illustrates (a) the separation resulting from this unsupervised classification between pancreatic cancer (with individual samples in grey) and controls (in black), as well as (b) the relative intensities of these 20 biomarkers in both populations.

1B. Logistic Regression Analysis

Receiver Operating Characteristic (ROC) analysis was performed on these 20 best FTICR biomarkers. Table 7 summarizes the resulting Areas Under the Curves (AUCs).

TABLE 7 List of FTICR biomarkers sorted by p-values with corresponding AUCs. Area Under Masses P-value the Curve 594.4863 9.91E−14 0.961 785.5913 1.39E−13 0.932 702.5709 2.39E−13 0.909 807.5734 5.49E−13 0.933 576.4751 5.61E−13 0.925 541.3134 6.66E−13 0.921 779.5405 2.66E−12 0.934 812.6774 3.81E−12 0.895 783.569 1.19E−11 0.906 596.5017 2.03E−11 0.932 803.5373 2.11E−11 0.924 724.5477 3.75E−11 0.878 519.3295 7.30E−11 0.899 757.556 1.04E−10 0.916 600.5117 2.61E−10 0.855 809.5796 2.67E−10 0.895 829.5516 2.82E−10 0.877 523.3661 3.97E−10 0.877 805.5549 6.17E−10 0.897 833.5864 9.05E−10 0.888

At least nine markers display AUC>0.90, which indicates an excellent specificity and sensitivity. FIG. 4 illustrates each ROC along with the distribution of sample values for the first six best biomarkers (p-value<E-12).

There are multiple ways of combining the best biomarkers in the perspective of obtaining a very high sensitivity and specificity with few of them. For example the combination of the six best biomarkers as classified by p-values displays an AUC of 0.985 (FIG. 5), with an optimal specificity and sensitivity pair of 92.5% and 88% respectively.

1C. Formula Prediction

Computational assignments of reasonable molecular formulae were performed for the 20 best biomarkers. The assignments were based on a series of mathematical and chemometric rules as previously described (24), which rely on high mass accuracy for precise prediction. The algorithm computes the number of carbons, hydrogens, oxygens, and other elements, based on their exact mass, which can be assigned to a detected accurate mass within defined constraints. Logical putative molecular formulae were computed in Table 8.

TABLE 8 Putative molecular formulae for the 20 best FTICR biomarkers. Ratio Analysis Detected pancreatic Mode Mass Putative formula P value cancer/control 1101 519.3295 C₂₆H₅₀NO₇P 7.30E−11 0.41 523.3661 C₂₆H₅₄NO₇P 3.97E−10 0.49 541.3134 C₂₈H₄₈NO₇P 6.66E−13 0.37 or C₂₆H₄₉NO₇PNa 702.5709 C₃₉H₇₉N₂O₆P 2.39E−13 0.47 724.5477 C₄₁H₇₇N₂O₆P 3.75E−11 0.49 or C₃₉H₇₈N₂O₆PNa 757.556 C₄₂H₈₀NO₈P 1.04E−10 0.27 779.5405 C₄₄H₇₈NO₈P 2.66E−12 0.26 783.569 C₄₄H₈₂NO₈P 1.19E−11 0.36 785.5913 C₄₄H₈₄NO₈P 1.39E−13 0.27 803.5373 C₄₈H₇₈NO₈P 2.11E−11 0.33 or C₄₄H₇₉NO₈PNa 805.5549 C₄₆H₈₀NO₈P 6.17E−10 0.35 807.5734 C₄₆H₈₂NO₈P 5.49E−13 0.28 809.5796 C₄₆H₈₄NO₈P 2.67E−10 0.43 812.6774 C₄₇H₉₃N₂O₈P 3.81E−12 0.54 829.5516 C₄₈H₈₀NO₈P 2.82E−10 0.41 or C₄₆H₈₁NO₈PNa 833.5864 C4₈H₈₄NO₈P 9.05E−10 0.43 or C₄₆H₈₅NO₈PNa 1202 576.4751 C₃₆H₆₄O₅ 5.61E−13 0.40 594.4863 C₃₆H₆₆O₆ 9.91E−14 0.31 596.5017 C₃₆H₆₈O₆ 2.03E−11 0.36 1203 600.5117 C₃₉H₆₈O₄ 2.61E−10 1.40

Four main families seem to emerge, three in 1101 analysis mode and one in 1202 analysis mode. In 1101 mode they are reminiscent of choline-related compounds, namely lysophosphatidylcholines for compounds in NO₇P, phosphatidylcholines for compounds in NO₈P, and sphingomyelins for compounds in N₂O₆P. The next step was the structural validation of these 16 putative choline-related compounds, the three compounds in C36 and the additional compound in 1203 mode.

HPLC-Coupled Tandem Mass Spectrometry

Tandem mass spectrometric fragmentation fingerprints were generated for the markers mentioned above.

2A. 1202/1204 Compounds in C36

Selected ethyl acetate extracts of serum from the control cohort used in the FTICR-MS work were re-analyzed using HPLC coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer in APCI negative ion mode (1202 mode) for the three C36 biomarkers, “576”, “594” and “596”. For a retention time around 25-27 minutes, the MS/MS and MS3 fragmentation data were dominated by peaks resulting from losses of H₂O (m/z 557, 575 and 577 respectively) and losses of two molecules of H₂O (m/z 539, 557 and 559 respectively), with smaller peaks corresponding to losses of CO₂ (m/z 531, 549 and 551 respectively) and losses of CO₂ and H₂O (m/z 513, 531 and 533) (Table 9; FIGS. 6 to 12).

TABLE 9 Fragmentation pattern of biomarkers “576”, “594” and “596” in negative APCI mode (with m/z 575, 593 and 595 respectively), with daughter ion relative abundance. Parent Parent Parent mass mass mass Predicted 576.5 Predicted 594.5 Predicted 596.5 formula C36H64O5 formula C36H66O6 formula C36H68O6 m/z575 m/z593 m/z595 Mass Intensity Mass Intensity Mass Intensity 495.4234 100 593.4734 100 279.2176 100 575.5086 100 575.4275 94 595.4591 86 513.4442 80 513.4442 65 315.2409 64 557.4564 80 371.3305 53 577.4549 55 539.4565 60 557.4476 53 515.4361 41 575.3825 60 315.2542 47 297.2472 36 97.0558 40 277.2144 41 559.452 36 403.3057 40 171.1025 35 595.6056 36 415.3021 40 201.101 35 281.228 27 459.3655 40 575.5266 35 313.2118 27 531.4755 40 279.2113 29 171.0829 23 71.0055 20 297.2407 24 576.4453 23 89.0176 20 513.5378 24 141.1259 18 101.0108 20 531.4495 24 577.5812 18 113.0104 20 557.5539 24 169.1396 14 119.0578 20 593.638 24 251.2339 14 123.0715 20 200.091 18 277.2081 14 125.0865 20 281.2217 18 373.3293 14 185.1142 20 313.2716 18 391.3588 14 197.1239 20 415.2715 18 594.507 14 205.193 20 433.3294 18 594.6352 14 251.2101 20 113.0862 12 125.0949 9 277.2081 20 139.1091 12 127.1136 9 279.2301 20 155.1033 12 153.1139 9 295.2963 20 195.1371 12 155.1126 9 297.2213 20 199.0942 12 207.214 9 371.2799 20 233.2058 12 239.2276 9 373.3873 20 251.2279 12 253.2247 9 387.3672 20 261.2057 12 261.2179 9 389.3049 20 263.2417 12 278.2338 9 417.3544 20 295.1996 12 295.2189 9 429.3153 20 311.1893 12 298.2186 9 431.3005 20 391.3737 12 372.3292 9 441.3348 20 403.3434 12 423.3793 9 445.3017 20 421.3739 12 497.4302 9 463.2347 20 495.4067 12 514.4141 9 529.4355 20 549.4484 12 515.5639 9 539.352 20 111.0599 6 516.4506 9 557.5893 20 125.0949 6 533.424 9 127.1051 6 558.4582 9 141.0992 6 559.6117 9 169.1103 6 595.6698 9 183.0976 6 115.0181 5 185.1039 6 143.099 5 221.1523 6 185.1091 5 283.2708 6 201.1223 5 289.2268 6 202.1455 5 309.3185 6 233.2287 5 331.3406 6 235.147 5 353.3364 6 239.0883 5 373.322 6 249.1502 5 389.3346 6 249.2509 5 401.2808 6 263.2417 5 417.3774 6 265.2183 5 446.3477 6 281.3413 5 451.3569 6 314.2888 5 453.4963 6 361.3032 5 514.4737 6 371.3305 5 549.5451 6 373.5324 5 559.0969 6 387.3451 5 564.3806 6 405.3583 5 568.1941 6 407.3632 5 576.3011 6 433.306 5 592.3951 6 438.3753 5 594.26 6 483.4343 5 594.4887 6 497.5474 5 531.4495 5 532.5142 5 533.554 5 533.6667 5 540.2675 5 541.4458 5 549.6155 5 551.5353 5 558.3518 5 560.3933 5 561.2821 5 577.7255 5 594.9372 5

Among FTICR biomarkers in Table 5, the presence of other compounds in 1202 mode with a mass differing from the masses above only by two or four suggested that a whole family may be altered in pancreatic cancer. We therefore performed the same analysis as above for 574.5, 578.5, 592.5 and 558.4, respectively predicted to have a formula of C₃₆H₆₂O₅, C₃₆H₆₆O₅, C₃₆H₆₀O₆ and C₃₆H₆₂O₄(Table 10; FIGS. 9 to 12).

TABLE 10 Fragmentation pattern of biomarkers “558”, “574”, “578” and “592” in negative APCI mode, with daughter ion relative abundance. Parent Parent Parent Parent mass mass mass mass Predicted 592.5 Predicted 558.5 Predicted 574.5 Predicted 578.5 formula C36H64O6 formula C36H62O4 formula C36H62O5 formula C36H66O5 m/z591 m/z 557 m/z573 m/z577 Mass Intensity Mass Intensity Mass Intensity Mass Intensity 591.3998 100 495.4401 100 573.3857 100 515.402 100 171.0927 75 539.3868 40 125.0991 80 497.4302 67 201.0903 75 557.4298 40 511.3968 80 533.4673 67 511.3543 75 111.0836 30 555.3937 80 541.4196 67 573.4127 75 539.5089 30 171.1025 40 559.4431 67 125.0907 50 279.2176 20 223.1101 40 577.464 67 223.1661 50 97.0632 10 277.1956 40 251.1982 33 255.2113 50 205.1823 10 279.2301 40 283.2393 33 279.1987 50 221.1467 10 457.3247 40 297.1955 33 295.206 50 373.3365 10 493.3789 40 405.4037 33 403.3358 50 494.5049 10 511.5665 40 515.5469 33 497.4637 50 495.5737 10 529.3751 40 576.4904 33 515.4105 50 513.4187 10 537.3752 40 529.4701 50 555.5264 40 555.4025 50 113.0782 20 559.4253 50 205.1823 20 573.5834 50 295.2447 20 591.6189 50 385.3239 20 111.0639 25 389.3346 20 113.0263 25 401.3484 20 127.0882 25 415.3709 20 203.1713 25 429.3309 20 275.1623 25 443.3555 20 277.2144 25 519.3887 20 297.2213 25 574.2218 20 313.245 25

Several classes of metabolites, including various forms of steroids (or bile acids), fatty acids and fat soluble vitamins theoretically fit these elemental compositions.

Preliminary Isolation of C36 Markers and NMR Analysis

Ethyl acetate extracts of commercial serum subjected to reverse phase flash column chromatography with a step gradient elution; acetonitrile-water 25:75 to 100% acetonitrile resulted in a fraction found to be very rich in two pancreatic cancer C36 markers (m/z 594 and 596) when analyzed by LC/MS and MS/MS. The proton nuclear magnetic resonance (¹H NMR) spectrum (FIG. 13) of this fraction showed resonances characteristic of compounds with condensed ring systems thought to be pregnane ring. These two markers are thought to have a steroidal backbone and may probably belong to a class of compounds known as bile acids.

2B. Putative Choline-Related Compounds

In table 6, 16 compounds showed putative formulas belonging to three choline-related families, namely lysophosphatidylcholines (LysoPC) for 519.3, 523.3, and 541.3, phosphatidylcholines (PtdCho) for 757.6, 779.5, 783.6, 785.6, 803.5, 805.6, 807.6, 809.6, 829.6 and 833.6, and sphingomyelins for 702.6, 724.5 and 812.7.

Selected aqueous extracts of serum from the control cohort used in the FTICR-MS work were re-analyzed using HPLC coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer in ESI positive ion mode (1101 mode). Multiple fragmentation patterns were observed for the three putative lysophosphatidylcholines (FIGS. 14 to 16).

TABLE 11 Fragmentation pattern of putative lysophosphatidylcholines in positive ESI mode, with daughter ion relative abundance. Accurate/ MS/MS Exact Parent ion Collision Mass (% intensity) Daughter ions (% intensity) Energy 519.3295 520 (6%) 283 (8%), 209 (3%), 184 (100%), 40 V 177 (3%), 175 (8%), 130 (11%), 125 (8%), 109 (6%), 104 (14%), 86 (11%) 523.3661 524 (20%) 506 (11%), 185 (3%), 184 (100%), 30 V 401 (62%), 86 (2%) 541.3134 542 (14%) 483 (88%), 439 (6%), 359 (8%), 40 V Na adduct 337 (22%), 177 (6%), 147 (72%), 421 (6%), 104 (100%), 86 (28%)

The compound with a mass of 519.3 is confirmed to be a lysophosphatidylcholine with a fatty acid moiety of C18:2, and the two different retention times correspond to two different subspecies: the lower time shows the fragmentation pattern of the 1-linoleoyl-sn-glycero-3-phosphocholine (FIG. 14a ) whereas the higher shows the fragmentation pattern of the 2-linoleoyl-sn-glycero-3-phosphocholine (FIG. 14b ).

The compound with a mass of 523.3 is confirmed to be a lysophosphatidylcholine with a fatty acid moiety of C18:0, and different retention times correspond to two different subspecies: the lower time shows the fragmentation pattern of the 2-stearoyl-sn-glycero-3-phosphocholine (FIG. 15a ) whereas the higher shows the fragmentation pattern of the 1-stearoyl-sn-glycero-3-phosphocholine (FIG. 15b ).

The compounds with a mass of 541.3 seem to be a mixture of the lysophosphatidylcholines with a fatty acid moiety of C20:5 and of the sodium adduct of the compounds with a mass of 519.3 above mentioned (FIG. 16). The lowest retention time shows indeed two fragmentation patterns corresponding to 1-eicosapentaenoyl-sn-glycero-3-phosphocholine (FIG. 16a ) and 2-eicosapentaenoyl-sn-glycero-3-phosphocholine (FIG. 16b ). The two higher retention times observed reflect the two retention times observed for 519.3, with the lower corresponding to the fragmentation pattern of the sodium adduct of the 1-linoleoyl-sn-glycero-3-phosphocholine (FIG. 16c ), and the higher corresponding to the fragmentation pattern of the sodium adduct of the 2-linoleoyl-sn-glycero-3-phosphocholine (FIG. 16d ).

In order to further validate the chemical family of these putative lysophosphatidylcholines, the same samples were run in aqueous negative ESI mode (Table 12).

TABLE 12 Fragmentation pattern of putative lysophosphatidylcholines in negative ESI mode, with daughter ion relative abundance. MS/MS Accurate/ Formic acid Exact adduct Collision Mass (% intensity) Daughter ions (% intensity) Energy 519.3295 564.3 (1%) 504 (5%), 279 (100%), 242 (2%), −35 V 224 (6%) 523.3661 568.3 (1%) 508 (14%), 283 (100%), 242 (2%), −35 V 224 (6%)

Selected aqueous extracts of serum from the control cohort used in the FTICR-MS work were re-analyzed using HPLC coupled to a Q-TOF mass spectrometer in ESI positive ion mode (1101 mode) for the putative PtdCho (Table 13).

TABLE 13 Fragmentation pattern of putative phosphatidylcholines in positive ESI mode, with daughter ion relative abundance. Accurate/ MS/MS Exact Parent ion Collision Mass (% intensity) Daughter ions (% intensity) Energy 757.5560 758 (47%) 184 (100%) 30 V 779.5405 780 (68%) 721 (7%), 712 (4%), 597 (4%), 30 V 184 (100%) 783.5690 784 (55%) 184 (100%) 30 V 785.5913 786 (66%) 184 (100%) 30 V 803.5373 804 26%) 745 (49%), 621 (100%), 599 (6%), 40 V (Na adduct) 313 (4%), 147 (17%) 805.5549 806 (95%) 747 (10%), 623 (6%), 184 (100%) 30 V 807.5734 808 (80%) 749 (5%), 624 (5%), 184 (100%) 30 V 809.5796 810 (100%) 751 (8%), 627 (6%), 184 (89%) 30 V 829.5516 830 (47%) 771 (53%), 647 (100%), 625 40 V (Na adduct) (10%), 147 (22%), 86 (7%) 833.5864 834 (96%) 775 (6%), 651 (3%), 415, (2%), 30 V 184 (100%)

Fragmentation pattern of all compounds seems restricted to one main fragment (m/z 184) for all masses, which likely corresponds to choline phosphate (FIGS. 17 to 20, 22 to 26), except for 803.5 (FIG. 21). The fragmentation pattern of 803.5 rather suggests the majority of the compounds at this mass to be the sodium adducts of 781.5566.

In order to confirm the chemical family of these putative phosphatidylcholines, the same samples were run in aqueous negative ESI mode (Table 14). Fragmentation patterns are shown in FIGS. 27 to 32 that show how to determine the PtdCho side chains.

TABLE 14 Fragmentation pattern of putative phosphatidylcholines in negative ESI mode, with daughter ion relative abundance. MS/MS Accurate/ Formic acid Exact adduct Collision Mass (% intensity) Daughter ions (% intensity) Energy 757.5560 802.5 (1%) 745 (9%), 480 (9%), 279 (100%), −35 V 255 (26%) 779.5405 824.5 (12%) 764 (100%), 480 (4%), 301 (30%), −35 V 255 (19%) 783.5690 828.6 (16%) 768 (100%), 480 (5%), 305 (15%), −35 V 279(9%), 255 (9%), 224 (2%) 785.5913 830.6 (1%) 770 (11%), 283 (22%), 281 (11%), −45 V 279 (100%) 803.5373 848.5 (1%) 788 (100%), 576 (8%), 508 (8%), −35 V 492 (8%), 474 (8%), 440 (16%), 301 (16%) 805.5549 850.6 (1%) 790 (33%), 255 (100%) −35 V 807.5734 852.6 (1%) 792 (32%), 508 (16%), 480 (12%), −45 V 329 (52%), 301 (100%), 283 (56%), 257 (48%), 255 (336%), 224 (16%), 203 42%) 809.5796 854.6 (5%) 794 (23%), 508 (12%), 378 (7%), −45 V 303 (100%), 283 (41%), 259 (17%), 242 (9%), 227 (7%), 205 (9%), 168 (7%) 829.5516 852.6 (1%) 792 (32%), 508 (16%), 480 (12%), −45 V 329 (52%), 301 (100%), 283 (56%), 257 (48%), 255 (336%), 224 (16%), 203 12%) 833.5864 878.6 (1%) 818 (33%), 508 (33%), 490 (33%), −35 V 327 (67%), 283 (100%)

Side chain combinations may be unique, such as in 757.6, corresponding to both PtdCho 16:0/18:2 and PtdCho 18:2/16:0 (FIG. 27), or multiple, such as in 807.6, corresponding to PtdCho 18:0/20:5, PtdCho 16:0/22:5 and PtdCho 18:1/20:4, all with the same chemical formula C₄₆H₈₂NO₈P (FIG. 32). Confirmed side chains for all PtdCho biomarkers are reported in Table 15.

TABLE 15 Assignment of side chains to PtdCho according to MS/MS data analysis Mass Formula Identity 757.556 C₄₂H₈₀NO₈P PtdCho16:0/18:2 PtdCho18:2/16:0 779.5405 C₄₄H₇₈NO₈P PtdCho18:3/18:2 PtdCho16:0/20:5 PtdCho20:5/16:0 PtdCho20:4/18:1 783.569 C₄₄H₈₂NO₈P PtdCho16:0/20:3 PtdCho18:1/18:2 PtdCho18:0/18:3 785.5913 C₄₄H₈₄NO₈P PtdCho18:0/18:2 PtdCho18:1/18:1 803.5373 C₄₆H₇₈NO₈P PtdCh020:5/18:2 PtdCho16:1/22:6 PtdCho22:6/16:1 805.5549 C₄₆H₈₀NO₈P PtdCho22:6/16:0 PtdCho18:2/20:4 807.5734 C₄₆H₈₂NO₈P PtdCho18:0/20:5 PtdCho16:0/22:5 PtdCho18:1/20:4 PtdCho22:5/16:0 809.5796 C₄₆H₈₄NO₈P PtdCho18:0/20:4 PtdCho18:1/20:3 PtdCho18:2/20:2 PtdCho16:0/22:4 827.5401 C₄₈H₇₈NO₈P Na adduct of 805.55 PtdCho18:3/22:6 829.5516 C4₈H₈₀NO₈P PtdCho18:2/22:6 Na adduct of 807.57 PtdCho18:3/22:5 833.5864 C₄₈H₈₄NO₈P PtdCho22:6/18:0 Na adduct of 811.6 PtdCho18:1/22:5 PtdCho16:0/24:6

The fragmentation pattern of the putative sphingomyelins confirmed the presence of a choline phosphate fragment as the major peak for 702.6 and 812.7, suggesting that these two compounds respectively are the common sphingomyelins SM(d18:1/16:0) and SM(d18:1/24:1(15Z)) with the sphingosine (18:1) as the sphingoid base (FIGS. 33 and 34). The fragmentation pattern of 724.5 suggests that the compound is the sodium adduct of 702.6 above mentioned (FIG. 35).

The sphingomyelin identity of these two compounds was confirmed by a further analysis in aqueous negative ESI mode, through the comparison between the serum compounds with a mass of 702.6 and 812.7 and the commercially available sphingomyelins SM(d18:1/16:0) and SM(d18:1/24:1(15Z)). The fragmentation pattern of the serum compound with a mass of 702.6 detected as a formic acid adduct in negative ESI mode (FIG. 36) is indeed identical to the fragmentation pattern of the synthetic SM(d18:1/16:0) (FIG. 37). Similarly, the fragmentation pattern of the serum compound with a mass of 812.7 detected as a formic acid adduct in negative ESI mode (FIG. 38) is identical to the fragmentation pattern of the synthetic SM(d18:1/24:1(15Z)) (FIG. 39).

2C. Other Compound

600.5117 compound in 1203 analysis mode was further analyzed by tandem mass spectrometry mass fragmentation. The fragmentation pattern, dominated by peaks at 545.5, 527.5 and 263.3, confirms that a compound with the molecular formula indicated in table 6 is present and can be classified as 1-alkenyl-2-acylglycerol with 18:2 at both side chains (FIG. 40).

Validation using Multiple Reaction Monitoring Methodology

Reduced levels of choline-related compounds and C36 biomarkers in the blood of pancreatic cancer patients were further confirmed using a tandem mass spectrometry approach (see methods) in the same populations. The approach is based upon the measurement of parent-daughter fragment ion combinations (referred to as multiple-reaction monitoring; MRM) for quantifying analytes.

3A. MRM for Lysophosphatidylcholines

A tandem-MS approach based upon multiple reaction monitoring was used to confirm differences in LysoPC levels between patients and controls using the same aqueous extracts as for the FTICR-MS analysis, in both positive and negative ElectroSpray Ionization modes (see methods for formulae and transitions). FIG. 41 reports the confirmation that the levels in the 3 lysophosphatidylcholines listed in Table 6 and in 20 additional LysoPC are significantly decreased in pancreatic cancer patients relative to controls. The lowest p-values among all LysoPC tested by MRM are obtained for LysoPC present in the 20 best FTICR biomarkers as could be expected, with the minimal value in positive ESI analysis mode, 2.69E-15, obtained for LysoPC 18:2, the second best putative LysoPC by FTICR. Overall, the significant decreases observed in 23 LysoPC suggest that the whole family is down-regulated in pancreatic cancer serum.

3B. MRM for PtdCho and Plasmenylphosphocholine (PlaCho)

The same aqueous extracts as for the FTICR-MS analysis were analyzed by a targeted method for 7 PtdCho out of the 10 listed in Table 6 and 6 additional PtdCho in positive analysis mode, and for 9 PtdCho out of the 10 listed in Table 6 and many additional PtdCho in negative analysis mode. FIGS. 42a and 42b report the confirmation that the serum levels of all PtdCho tested in both positive and negative ESI analysis modes are significantly decreased in pancreatic cancer patients relative to controls. The best putative PtdCho among FTICR best biomarkers, “785.6”, is also the best PtdCho among all tested by MRM in positive ESI analysis mode, with a p-value of 5.77E-18. It is interesting to note that all PtdCho tested are decreased in pancreatic cancer serum independently of their side chains, with a maximal p-value of 5.31E-10 in positive ESI analysis mode, demonstrating that the whole phosphatidylcholine family is collectively down-regulated in pancreatic cancer serum.

The decrease in PtdCho family incited us to assess the levels of their vinyl ether counterparts, plasmenylphosphocholines (PlsCho), in the same samples. FIG. 42c reports that the serum levels of all PlsCho tested in positive Electrospray Ionization analysis mode are very significantly decreased in pancreatic cancer patients relative to controls. PlsCho with a mass of 793.6, which likely is PlsCho 18:0/20:4, shows the lowest p-value, 3.9E-17.

3D. MRM for Sphingomyelins

A tandem-MS approach based upon multiple reaction monitoring was developed to confirm differences in sphingomyelin levels between patients and controls using the same aqueous extracts as for the FTICR-MS analysis. FIG. 43 reports that the serum levels of the five sphingomyelins tested (including the two identified by FTICR analysis, SM(dl 8:1/16:0) and SM(d18:1/24:1(15Z)) are very significantly decreased in pancreatic cancer patients relative to controls. SM(d18:1/24:0), which had not been detected by FTICR, shows the strongest decrease with a p-value of 7.81E-15.

3D. MRM for C36 Biomarkers

A tandem-MS approach based upon multiple reaction monitoring was developed to confirm differences in C36 biomarker levels between patients and controls using the same ethyl acetate extracts as for the FTICR-MS analysis. As explained in 2A, among all masses listed in Table 5, several seemed to belong to a same family in C36, only differing by an H₂O molecule or the number of unsaturations, and the tandem-MS method was extended to the whole “C36 family” (see methods for formulae and transitions).

FIG. 44 reports the confirmation that the levels in the seven C36 markers tested are significantly decreased in pancreatic cancer patients relative to controls. The best putative C36 marker among all FTICR biomarkers (which is also the best biomarker of pancreatic cancer), “594”, is also the best biomarker among all C36 tested by MRM, with a p-value of 1.42E-11. Again, it is interesting to note that as a whole family, the C36 markers seem down-regulated in pancreatic cancer serum.

Disease Stage Analysis

Information regarding disease progression status was included. It was therefore determined whether there were a correlation between disease progression and biomarker decrease. MRM data for the 3 LysoPC, 7 PtdCho and 3 C36 markers of interest were re-analyzed according to cancer stage (FIG. 45). This preliminary study on a small amount of patients per stage does not seem to indicate any trends.

Chemoradiation Therapy Effects on Biomarkers

Information regarding chemoradiation therapy status was included. It was therefore determined whether there was a correlation between this kind of therapy and biomarker decrease. MRM data for the 3 LysoPC, 7 PtdCho and 3 C36 markers of interest were re-analyzed according to therapy status (FIG. 46). This preliminary study on a small amount of patients seems to indicate that there is no effect of chemoradiation therapy on biomarkers.

Discussion

We have performed a comprehensive non-targeted metabolomic profiling of pancreatic cancer serum samples and have identified a very strong signature of this cancer as illustrated by most AUCs above 0.90. The families of markers identified by FTICR as discriminating were validated by targeted analysis. Four families have been identified whose decrease is associated to pancreatic cancer: phosphatidylcholines, lysophosphatidylcholines, sphingomyelins and C36 markers that may be steroidal-like compounds.

Lysophosphatidylcholines 18:2, 18:3 and 20:5 show the strongest decrease of all LysoPC tested. All 27 PtdCho tested (with nine included in the top list of Table 6) show significantly decreased levels in pancreatic cancer patients relative to controls (FIG. 42a,b ). Most of the 10 PtdCho in Table 8 are predicted or shown to have 18:2, 20:5 or 22:5 as one of the two side chains, as seen in Table 15. In summary, phosphatidylcholines and lysophosphatidylcholines that contain 18:2, 18:3, 20:5 and in a lesser extent, 22:5, show the strongest decrease.

The presence of sphingomyelins among the best biomarkers is extremely interesting. The role of sphingomyelin in cell death, growth and differentiation, and therefore in cancer, is well documented (25, 26) and cancer therapeutics targeted to their signaling pathways give very promising preliminary results (27, 28). For example, sphingomyelin addition to pancreatic cancer cell lines has been shown to drastically enhance chemosensitivity to anticancer agents, presumably by redirecting the cell to enter the apoptotic pathway (29).

Without wishing to be bound in any way by theory, the alteration observed in both phosphatidylcholines and sphingomyelins suggests a role for choline kinase; this cytosolic enzyme is indeed important for the generation of both species and subsequently for cell division (11). The involvement of the choline kinase during tumorigenesis (mediated by Ras effectors serine/threonine kinase (Raf-1), Ral-GDS and PI3K) and the success of its specific inhibitors in antitumoral activity make this kinase a very attractive target in cancer (11, 30). The present results therefore suggest an involvement of choline kinase in pancreatic carcinogenesis.

The C36 markers described herein have not, to our knowledge, yet been associated to pancreatic cancer. Preliminary NMR studies suggest that these compounds may be steroidal-like or conjugated bile acids. This is very interesting since bile acids are emerging as an important family in cancers of the gastrointestinal tract (31). Mechanistically speaking, although without wishing to be bound by theory, there is a complex balance in the bile between bile salts and phospholipids; the reduced levels in phosphatidylcholines observed in pancreatic cancer may be caused by a reduced export into bile, which could be reflective of MDR3 gene polymorphisms (20). An unbalance observed between phosphatidylcholines and bile acids may therefore reflect some genetic alterations underlying carcinogenesis.

A major effect of clinical variables on the alterations of biomarkers has not been identified on the whole pancreatic cancer population. Disease stages do not seem to affect the decrease in biomarkers. The observation that there are no stage effects suggests that the metabolic deficiency may precede the development of pancreatic cancer, and therefore supports the utility as an early detection risk screening method. A chemoradiation therapy effect on biomarkers was also not observed, suggesting that this therapy does not affect the underlying mechanism of pancreatic cancer, a normalization of biomarkers after treatment would therefore be a good efficacy indicator of new therapeutics.

Statistical analysis revealed how discriminating a few biomarkers could be between pancreatic cancer and healthy controls. For example, the six FTICR best biomarkers all present with a p-value lower than 1E-12 and individual AUCs above 0.90. They have been afterwards identified as most likely being a lysophosphatidylcholine, a sphingomyelin, two phosphatidylcholines and two C36 markers (one being the best biomarker, “594”, with p=9.9E-14 and the highest AUC). When these markers are combined, the AUC reaches 0.985, with a specificity of 92.5% and a sensitivity of 88%, illustrating how a blood draw can be a powerful diagnostic tool in pancreatic cancer.

In summary, we have identified a metabolic dysregulation specific to pancreatic cancer. The characteristic decrease in two main metabolite families, glycerophosphocholine-related compounds (sub grouped in three subfamilies) and previously uncharacterized C36 markers. These metabolites represent useful biomarkers for sensitive and specific detection of pancreatic cancer, which remains the most dreaded cancer because of its extremely low survival rate. The described diagnostic methods, when conducted in conjunction with therapeutic optimization steps, may also be used to design more efficacious drug therapies for the disease.

One or more currently preferred embodiments have been described by way of example. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

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World J Gastroenterol 15: 3329-3340. 

1.-18. (canceled)
 19. An analytical method for diagnosing pancreatic cancer in a patient, comprising the steps of: a) performing a mass spectrometry assay on at least one blood sample from said patient using a Fourier transform ion cyclotron resonance, time of flight, magnetic sector, quadrupole or triple quadrupole mass spectrometer to obtain a collision induced dissociation (CID) MS/MS fragmentation pattern for one or more than one metabolite marker and to obtain quantifying data for said one or more than one metabolite marker; b) generating a result of said mass spectrometry assay, said result comprising at least the presence of a decrease in the level of said one or more than one metabolite marker in said blood sample based on a comparison of said quantifying data for said one or more than one metabolite marker to corresponding data obtained for one or more than one reference blood sample; and c) assigning the patient as having pancreatic cancer based on the decrease in the level of said one or more than one metabolite marker in said blood sample, wherein the one or more than one metabolite marker is: lysophosphatidylcholine LysoPC 20:5, having a molecular formula of C₂₈H₄₈NO₇P and being characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas comprising the following daughter ions: 542.3 and 184.2 in positive ionization mode, 586.3 and 526.3 in negative ionization mode, and 586.3 and 301.2 in negative ionization mode; or a phosphatidylcholine having a molecular formula of C₄₄H₈₄NO₈P or C₄₆H₈₂NO₈P, the phosphatidylcholine having the molecular formula of C₄₄H₈₄NO₈P being characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas comprising the following daughter ions: 786.6 and 184.2 in positive ionization mode, 830.6, 770.6 and 279.2 for PtdCho 18:0/18:2 in negative ionization mode, 830.6, 770.6 and 283.2 for PtdCho 18:2/18:0 in negative ionization mode, and 830.6, 770.6 and 281.2 for PtdCho 18:1/18:1 in negative ionization mode, and the metabolite having the molecular formula of C₄₆H₈₂NO₈P being characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas comprising the following daughter ions: 808.6 and 184.2 in positive ionization mode, 852.6, 792.6 and 301.3 for PtdCho 18:0/20:5 in negative ionization mode, 852.6, 792.6 and 329.3 for PtdCho 16:0/22:5 in negative ionization mode, 852.6, 792.6 and 303.2 for PtdCho 18:1/20:4 in negative ionization mode, and 852.6, 792.6 and 255.2 for PtdCho 22:5/16:0 in negative ionization mode; or a sphingomyelin having a molecular formula of C₃₉H₇₉N₂O₆P and being characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas comprising the following daughter ions: 703.6 and 184.2 in positive ionization mode and 747.6, 687.6 and 168.1 in negative ionization mode; or has the molecular formula of C₃₆H₆₄O₅ and is characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas and analyzed under negative ionization comprising the following daughter ions: 575.5, 513.5, 557.5, 539.5, 531.5, 499.5, 495.5, 459.4, 417.4, 415.3, 413.3, 403.3, 295.2, 279.2, 260.2, 251.2, 197.9, 119.4, 113.1, and 97.0; or a combination thereof.
 20. The method of claim 19, wherein the one or more than one metabolite marker is a combination of metabolite markers that further comprises a marker having the molecular formula of C₃₆H₆₆O₆ and being characterized by a CID MS/MS fragmentation pattern using N₂ as collision gas and analyzed under negative ionization comprising the following daughter ions: 593.5, 557.5, 575.4, 549.4, 531.5, 513.4, 495.4, 433.3, 421.4, 415.2, 391.4, 371.3, 315.3, 311.1, 297.2, 281.2, 277.2, 251.2, 201.1, 195.3, 171.1, 139.1 and 133.5.
 21. The method of claim 19, wherein the one or more than one metabolite marker is the lysophosphatidylcholine LysoPC 20:5, having a molecular formula of C₂₈H₄₈NO₇P.
 22. The method of claim 19, wherein the one or more than one metabolite marker is the phosphatidylcholine having a molecular formula of C₄₄H₈₄NO₈P.
 23. The method of claim 19, wherein the one or more than one metabolite marker is the phosphatidylcholine having a molecular formula of C₄₆H₈₂NO₈P.
 24. The method of claim 19, wherein the one or more than one metabolite marker is the sphingomyelin having a molecular formula of C₃₉H₇₉N₂O₆P.
 25. The method of claim 19, wherein the one or more than one metabolite marker is C₃₆H₆₄O₅.
 26. The method of claim 19, wherein the mass spectrometer is equipped with a chromatographic system.
 27. The method of claim 19, wherein the blood sample is a blood serum sample.
 28. The method of claim 19, wherein a liquid/liquid extraction is performed on the blood sample whereby non-polar metabolites are dissolved in an organic solvent and polar metabolites are dissolved in an aqueous solvent.
 29. The method of claim 28, wherein the extracted samples are analyzed by: positive or negative electrospray ionization, positive or negative atmospheric pressure chemical ionization, or combinations thereof; by MS/MS transition; or by extracted ion current (EIC) chromatography and MS/MS transition.
 30. The method of claim 19, wherein said one or more than one reference blood sample is from one or more pancreatic cancer-negative humans.
 31. The method of claim 19, further comprising: performing a mass spectrometry assay on a blood sample from said patient to obtain quantifying data for one or more than one internal standard molecule; and obtaining a ratio for each of the levels of said one or more than one metabolite marker to the level obtained for the one or more than one internal standard molecule; wherein step (b) comprises generating said result based on a comparison of each ratio to one or more corresponding ratios obtained for the one or more than one reference sample. 